rat anti mouse cd86 pe  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 85

    Structured Review

    Thermo Fisher rat anti mouse cd86 pe
    Muscle damage markers in serum and inflammation in gastrocnemius muscle are decreased in 3-month-old dystrophic mice lacking miR-378. ( A ) Lower serum activity of LDH in mdx animals lacking miR-378; activity assay; n = 10–14/group. ( B ) Increased serum CK activity in mdx mice with a tendency to be decreased by miR-378-KO, activity assay; n = 11–13/group. ( C ) Necrosis assessment by immunofluorescent staining of IgM and IgG (green) binding and its calculation indicating no differences between groups; n = 9–10/group. Scale bar: 100 μm. ( D ) Representative pictures of H E staining of gastrocnemius muscle with semiquantitative analysis of inflammation extent showing a tendency in decreased inflammatory cell infiltration in dKO mice; microscopic assessment using Nikon Eclipse microscope. Scale bar: 100 μm; n = 4–6/group. ( E ) Decreased number of WBC in the peripheral blood in dKO mice; blood cell count; n = 5–6/group. ( F–J ) The analysis of inflammatory cells in hind limb muscles with special emphasis on macrophage subpopulations; flow cytometry analysis calculated as the percentage of Hoechst + cells; n = 5/group. The percentage of CD45 + cells ( F ), macrophages (CD45 + F4/80 + CD11b + cells) ( G ), M1-like macrophages (CD45 + F4/80 + CD11b + MHCII hi CD206 lo cells) ( H ), M2-like macrophages (CD45 + F4/80 + CD11b + MHCII lo CD206 hi cells) ( I ), and eosinophils (CD45 + F4/80 + <t>CD86</t> + cells) ( J ) showing significant decrease in dKO mice. ( K ) The decreased HO-1 protein level in dKO animals assessed by Western blot; GAPDH used as loading control. Representative result of 2 independent experiments; n = 4–5/group. Data are presented as mean ± SEM. * P
    Rat Anti Mouse Cd86 Pe, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 26717 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rat anti mouse cd86 pe/product/Thermo Fisher
    Average 85 stars, based on 26717 article reviews
    Price from $9.99 to $1999.99
    rat anti mouse cd86 pe - by Bioz Stars, 2020-09
    85/100 stars

    Images

    1) Product Images from "Lack of miR-378 attenuates muscular dystrophy in mdx mice"

    Article Title: Lack of miR-378 attenuates muscular dystrophy in mdx mice

    Journal: JCI Insight

    doi: 10.1172/jci.insight.135576

    Muscle damage markers in serum and inflammation in gastrocnemius muscle are decreased in 3-month-old dystrophic mice lacking miR-378. ( A ) Lower serum activity of LDH in mdx animals lacking miR-378; activity assay; n = 10–14/group. ( B ) Increased serum CK activity in mdx mice with a tendency to be decreased by miR-378-KO, activity assay; n = 11–13/group. ( C ) Necrosis assessment by immunofluorescent staining of IgM and IgG (green) binding and its calculation indicating no differences between groups; n = 9–10/group. Scale bar: 100 μm. ( D ) Representative pictures of H E staining of gastrocnemius muscle with semiquantitative analysis of inflammation extent showing a tendency in decreased inflammatory cell infiltration in dKO mice; microscopic assessment using Nikon Eclipse microscope. Scale bar: 100 μm; n = 4–6/group. ( E ) Decreased number of WBC in the peripheral blood in dKO mice; blood cell count; n = 5–6/group. ( F–J ) The analysis of inflammatory cells in hind limb muscles with special emphasis on macrophage subpopulations; flow cytometry analysis calculated as the percentage of Hoechst + cells; n = 5/group. The percentage of CD45 + cells ( F ), macrophages (CD45 + F4/80 + CD11b + cells) ( G ), M1-like macrophages (CD45 + F4/80 + CD11b + MHCII hi CD206 lo cells) ( H ), M2-like macrophages (CD45 + F4/80 + CD11b + MHCII lo CD206 hi cells) ( I ), and eosinophils (CD45 + F4/80 + CD86 + cells) ( J ) showing significant decrease in dKO mice. ( K ) The decreased HO-1 protein level in dKO animals assessed by Western blot; GAPDH used as loading control. Representative result of 2 independent experiments; n = 4–5/group. Data are presented as mean ± SEM. * P
    Figure Legend Snippet: Muscle damage markers in serum and inflammation in gastrocnemius muscle are decreased in 3-month-old dystrophic mice lacking miR-378. ( A ) Lower serum activity of LDH in mdx animals lacking miR-378; activity assay; n = 10–14/group. ( B ) Increased serum CK activity in mdx mice with a tendency to be decreased by miR-378-KO, activity assay; n = 11–13/group. ( C ) Necrosis assessment by immunofluorescent staining of IgM and IgG (green) binding and its calculation indicating no differences between groups; n = 9–10/group. Scale bar: 100 μm. ( D ) Representative pictures of H E staining of gastrocnemius muscle with semiquantitative analysis of inflammation extent showing a tendency in decreased inflammatory cell infiltration in dKO mice; microscopic assessment using Nikon Eclipse microscope. Scale bar: 100 μm; n = 4–6/group. ( E ) Decreased number of WBC in the peripheral blood in dKO mice; blood cell count; n = 5–6/group. ( F–J ) The analysis of inflammatory cells in hind limb muscles with special emphasis on macrophage subpopulations; flow cytometry analysis calculated as the percentage of Hoechst + cells; n = 5/group. The percentage of CD45 + cells ( F ), macrophages (CD45 + F4/80 + CD11b + cells) ( G ), M1-like macrophages (CD45 + F4/80 + CD11b + MHCII hi CD206 lo cells) ( H ), M2-like macrophages (CD45 + F4/80 + CD11b + MHCII lo CD206 hi cells) ( I ), and eosinophils (CD45 + F4/80 + CD86 + cells) ( J ) showing significant decrease in dKO mice. ( K ) The decreased HO-1 protein level in dKO animals assessed by Western blot; GAPDH used as loading control. Representative result of 2 independent experiments; n = 4–5/group. Data are presented as mean ± SEM. * P

    Techniques Used: Mouse Assay, Activity Assay, Staining, Binding Assay, Microscopy, Cell Counting, Flow Cytometry, Western Blot

    2) Product Images from "Deciphering hepatocellular responses to metabolic and oncogenic stress"

    Article Title: Deciphering hepatocellular responses to metabolic and oncogenic stress

    Journal: Journal of biological methods

    doi: 10.14440/jbm.2015.77

    CD45+ cells of the liver NPF are enriched for F4/80+ tissue-resident MΦ
    Figure Legend Snippet: CD45+ cells of the liver NPF are enriched for F4/80+ tissue-resident MΦ

    Techniques Used:

    3) Product Images from "Glial cell-derived neuroregulators control type 3 innate lymphoid cells and gut defence"

    Article Title: Glial cell-derived neuroregulators control type 3 innate lymphoid cells and gut defence

    Journal: Nature

    doi: 10.1038/nature18644

    Enteric homeostasis in steady-state Ret Δ mice. a , Rorgt -Cre mice were bread to Rosa26 YFP . Analysis of Rosa26/YFP expression in gut ILC3 from Rorgt -Cre. Rosa26 YFP mice. b , Number of Peyer’s patches (PP). Ret fl n=10; Ret Δ n=10. c , T cell derived IL-22 in Ret Δ mice and their WT littermate controls. Ret fl n=11; Ret Δ n=11. d , γδ T cell derived IL-22 in Ret Δ mice and their WT littermate controls. Ret fl n=4; Ret Δ n=4. e , Intestinal villus and crypt morphology. Ret fl n=6; Ret Δ n=6. f , Epithelial cell proliferation. Ret fl n=5; Ret Δ n=5. g , Intestinal paracellular permeability measured by Dextran-Fitc in the plasma. Ret fl n=5; Ret Δ n=5. h , Tissue repair genes in Ret Δ intestinal epithelium in comparison to their WT littermate controls. n=8. i , Reactivity genes in Ret MEN2B mice treated with anti-IL-22 blocking antibodies in comparison to Ret MEN2B intestinal epithelium. Ret MEN2B n=4; Ret MEN2B + anti-IL-22 n=4. Data are representative of 3 independent experiments. Error bars show s.e.m. ns not significant.
    Figure Legend Snippet: Enteric homeostasis in steady-state Ret Δ mice. a , Rorgt -Cre mice were bread to Rosa26 YFP . Analysis of Rosa26/YFP expression in gut ILC3 from Rorgt -Cre. Rosa26 YFP mice. b , Number of Peyer’s patches (PP). Ret fl n=10; Ret Δ n=10. c , T cell derived IL-22 in Ret Δ mice and their WT littermate controls. Ret fl n=11; Ret Δ n=11. d , γδ T cell derived IL-22 in Ret Δ mice and their WT littermate controls. Ret fl n=4; Ret Δ n=4. e , Intestinal villus and crypt morphology. Ret fl n=6; Ret Δ n=6. f , Epithelial cell proliferation. Ret fl n=5; Ret Δ n=5. g , Intestinal paracellular permeability measured by Dextran-Fitc in the plasma. Ret fl n=5; Ret Δ n=5. h , Tissue repair genes in Ret Δ intestinal epithelium in comparison to their WT littermate controls. n=8. i , Reactivity genes in Ret MEN2B mice treated with anti-IL-22 blocking antibodies in comparison to Ret MEN2B intestinal epithelium. Ret MEN2B n=4; Ret MEN2B + anti-IL-22 n=4. Data are representative of 3 independent experiments. Error bars show s.e.m. ns not significant.

    Techniques Used: Mouse Assay, Expressing, Derivative Assay, Permeability, Blocking Assay

    T cell-derived IL-22 and IL-17 in Ret GFP chimeras and Ret MEN2B mice. a , T cell derived IL-17 in Ret GFP chimeras. Ret WT/GFP n=25; Ret GFP/GFP n=22. b , T cell derived IL-22 and IL17 in the intestine of Ret MEN2B mice and their WT littermate controls. Ret WT n=7; Ret MEN2B n=7. Data are representative of 4 independent experiments. Error bars show s.e.m. ns not significant.
    Figure Legend Snippet: T cell-derived IL-22 and IL-17 in Ret GFP chimeras and Ret MEN2B mice. a , T cell derived IL-17 in Ret GFP chimeras. Ret WT/GFP n=25; Ret GFP/GFP n=22. b , T cell derived IL-22 and IL17 in the intestine of Ret MEN2B mice and their WT littermate controls. Ret WT n=7; Ret MEN2B n=7. Data are representative of 4 independent experiments. Error bars show s.e.m. ns not significant.

    Techniques Used: Derivative Assay, Mouse Assay

    ILC3-intrinsic RET signals regulate gut defence. a , ILC3-derived cytokines. n=11. b , Ret Δ and Ret MEN2B mice compared to their WT littermate controls. n=7. c-f , DSS treatment . Ret fl n=8; Ret Δ n=8. c , Histopathology. d , Inflammation score and colon length. e , Innate IL-22. f , Bacterial translocation. g-j , DSS treatment. Ret WT n=8; Ret MEN2B n=8. g , Histopathology. h , Inflammation score and colon length. i , Innate IL-22. j , Bacterial translocation. k-n , C. rodentium infection. Rag1 -/- . Ret fl n=15; Rag1 -/- . Ret Δ n=17. k , Histopathology. l , Inflammation score and colon length. m , Innate IL-22. n , Infection burden. Scale bars: 200µm. Data are representative of 4 independent experiments. Error bars show s.e.m. *P
    Figure Legend Snippet: ILC3-intrinsic RET signals regulate gut defence. a , ILC3-derived cytokines. n=11. b , Ret Δ and Ret MEN2B mice compared to their WT littermate controls. n=7. c-f , DSS treatment . Ret fl n=8; Ret Δ n=8. c , Histopathology. d , Inflammation score and colon length. e , Innate IL-22. f , Bacterial translocation. g-j , DSS treatment. Ret WT n=8; Ret MEN2B n=8. g , Histopathology. h , Inflammation score and colon length. i , Innate IL-22. j , Bacterial translocation. k-n , C. rodentium infection. Rag1 -/- . Ret fl n=15; Rag1 -/- . Ret Δ n=17. k , Histopathology. l , Inflammation score and colon length. m , Innate IL-22. n , Infection burden. Scale bars: 200µm. Data are representative of 4 independent experiments. Error bars show s.e.m. *P

    Techniques Used: Derivative Assay, Mouse Assay, Histopathology, Translocation Assay, Infection

    Glial cell sensing via MYD88 signals. a-c , Intestinal glial cells were purified by flow cytometry. a , Germ-free (GF) and their respective Specific Pathogen Free (SPF) controls. n=3. b , Myd88 -/- and their respective WT littermate controls. n=3. c , Gfap- Cre .Myd88 Δ and their littermate controls ( Myd88 fl ). n=3. d , Total lamina propria cells of Gfap- Cre .Myd88 Δ and their littermate controls ( Myd88 fl ). n=6. e-h , Citrobacter rodentium infection of Gfap- Cre .Myd88 Δ mice and their littermate controls ( Myd88 fl ). n=6. e , Innate IL-22. f , Citrobacter rodentium translocation. g , Infection burden. h , Weight loss. Data are representative of 3 independent experiments. Error bars show s.e.m. *P
    Figure Legend Snippet: Glial cell sensing via MYD88 signals. a-c , Intestinal glial cells were purified by flow cytometry. a , Germ-free (GF) and their respective Specific Pathogen Free (SPF) controls. n=3. b , Myd88 -/- and their respective WT littermate controls. n=3. c , Gfap- Cre .Myd88 Δ and their littermate controls ( Myd88 fl ). n=3. d , Total lamina propria cells of Gfap- Cre .Myd88 Δ and their littermate controls ( Myd88 fl ). n=6. e-h , Citrobacter rodentium infection of Gfap- Cre .Myd88 Δ mice and their littermate controls ( Myd88 fl ). n=6. e , Innate IL-22. f , Citrobacter rodentium translocation. g , Infection burden. h , Weight loss. Data are representative of 3 independent experiments. Error bars show s.e.m. *P

    Techniques Used: Purification, Flow Cytometry, Cytometry, Infection, Mouse Assay, Translocation Assay

    Glial cells set GFL expression and innate IL-22, via MYD88-dependent sensing of the microenvironment. a , Weighted Unifrac PCoA analysis and genus-level comparisons from co-housed Ret fl (white circles) and Ret Δ (black circles) littermates. n=5. Purple: Unclassified S24-7 ; Red: Bacteroides ; Green: Sutterella ; Blue: Unclassified Clostridiales ; Grey: Other. b-d , DSS treatment of colonised germ-free (GF) mice. n=5. b , Histopathology. c , Inflammation score. d , Innate IL-22. e , Innate IL-22 after antibiotic treatment. n=8. f , Ret GFP . Gfap -Cre. Rosa26 RFP mice. Green: RET/GFP; Red: GFAP/RFP. g,h , Glial cell activation with TLR2, TLR4, IL-1 β receptor and IL-33 receptor ligands. n=6. i , TLR ligands, IL-1β and IL-33 activation of co-cultured ILC3 with WT (white bars) or Myd88 -/- glial cells (black bars). n=6. j-m , DSS treatment of Gfap- Cre .Myd88 Δ mice. n=12. j , Histopathology. k , Inflammation score and colon length. l , Innate IL-22. m , Body weight. Scale bars: 200µm (b, j); 10µm (f). Data are representative of 3-4 independent experiments. Error bars show s.e.m. *P
    Figure Legend Snippet: Glial cells set GFL expression and innate IL-22, via MYD88-dependent sensing of the microenvironment. a , Weighted Unifrac PCoA analysis and genus-level comparisons from co-housed Ret fl (white circles) and Ret Δ (black circles) littermates. n=5. Purple: Unclassified S24-7 ; Red: Bacteroides ; Green: Sutterella ; Blue: Unclassified Clostridiales ; Grey: Other. b-d , DSS treatment of colonised germ-free (GF) mice. n=5. b , Histopathology. c , Inflammation score. d , Innate IL-22. e , Innate IL-22 after antibiotic treatment. n=8. f , Ret GFP . Gfap -Cre. Rosa26 RFP mice. Green: RET/GFP; Red: GFAP/RFP. g,h , Glial cell activation with TLR2, TLR4, IL-1 β receptor and IL-33 receptor ligands. n=6. i , TLR ligands, IL-1β and IL-33 activation of co-cultured ILC3 with WT (white bars) or Myd88 -/- glial cells (black bars). n=6. j-m , DSS treatment of Gfap- Cre .Myd88 Δ mice. n=12. j , Histopathology. k , Inflammation score and colon length. l , Innate IL-22. m , Body weight. Scale bars: 200µm (b, j); 10µm (f). Data are representative of 3-4 independent experiments. Error bars show s.e.m. *P

    Techniques Used: Expressing, Mouse Assay, Histopathology, Activation Assay, Cell Culture

    A novel glial-ILC3-epithelial cell unit orchestrated by neurotrophic factors. Lamina propria glial cells sense microenvironmental products, that control neurotrophic factor expression. Glial-derived neurotrophic factors operate in an ILC3-intrinsic manner by activating the tyrosine kinase RET, which directly regulates innate IL-22 downstream of a p38 MAPK/ERK-AKT cascade and STAT3 phosphorylation. GFL induced innate IL-22 acts on epithelial cells to induce reactivity gene expression (CBP: Commensal bacterial products; AMP: antimicrobial peptides; Muc: mucins). Thus, neurotrophic factors are the molecular link between glial cell sensing, innate IL-22 production and intestinal epithelial barrier defence.
    Figure Legend Snippet: A novel glial-ILC3-epithelial cell unit orchestrated by neurotrophic factors. Lamina propria glial cells sense microenvironmental products, that control neurotrophic factor expression. Glial-derived neurotrophic factors operate in an ILC3-intrinsic manner by activating the tyrosine kinase RET, which directly regulates innate IL-22 downstream of a p38 MAPK/ERK-AKT cascade and STAT3 phosphorylation. GFL induced innate IL-22 acts on epithelial cells to induce reactivity gene expression (CBP: Commensal bacterial products; AMP: antimicrobial peptides; Muc: mucins). Thus, neurotrophic factors are the molecular link between glial cell sensing, innate IL-22 production and intestinal epithelial barrier defence.

    Techniques Used: Expressing, Derivative Assay

    Citrobacter rodentium infection in Ret Δ mice. a , C. rodentium translocation to the liver of Rag1 -/- . Ret Δ and their Rag1 -/- . Ret fl littermate controls at day 6 post-infection. n=15. b , MacConkey plates of liver cell suspensions from Rag1 -/- . Ret Δ and their Rag1 -/- . Ret fl littermate controls at day 6 after C. rodentium infection. c , Whole-body imaging of Rag1 -/- . Ret Δ and their Rag1 -/- . Ret fl littermate controls at day 6 after luciferase-expressing C. rodentium infection. d , Epithelial reactivity gene expression in C. rodentium infected Rag1 -/- . Ret Δ mice and their Rag1 -/- . Ret fl littermate controls. Rag1 -/- . Ret fl n=15; Rag1 -/- . Ret Δ n=17. e , Weight loss in C. rodentium infected Rag1 -/- . Ret Δ mice and their Rag1 -/- . Ret fl littermate controls. Rag1 -/- . Ret fl n=8; Rag1 -/- . Ret Δ n=8. f , Survival curves in C. rodentium infected Rag1 -/- . Ret Δ mice and their Rag1 -/- . Ret fl littermate controls. Rag1 -/- . Ret fl n=8; Rag1 -/- . Ret Δ n=8. g , C. rodentium translocation to the liver of Ret Δ and their Ret fl littermate controls at day 6 post-infection. n=6. h , MacConkey plates of liver cell suspensions from Ret Δ and their Ret fl littermate controls at day 6 after C. rodentium infection. i , Whole-body imaging of Ret Δ and their Ret fl littermate controls at day 6 after luciferase-expressing C. rodentium infection. j , C. rodentium infection burden. Ret fl n=8; Ret Δ n=8. k , Innate IL-22 in in C. rodentium infected Ret Δ mice and their Ret fl littermate controls. Ret fl n=8; Ret Δ n=8. Data are representative of 3-4 independent experiments. Error bars show s.e.m. ns not significant. Error bars show s.e.m. *P
    Figure Legend Snippet: Citrobacter rodentium infection in Ret Δ mice. a , C. rodentium translocation to the liver of Rag1 -/- . Ret Δ and their Rag1 -/- . Ret fl littermate controls at day 6 post-infection. n=15. b , MacConkey plates of liver cell suspensions from Rag1 -/- . Ret Δ and their Rag1 -/- . Ret fl littermate controls at day 6 after C. rodentium infection. c , Whole-body imaging of Rag1 -/- . Ret Δ and their Rag1 -/- . Ret fl littermate controls at day 6 after luciferase-expressing C. rodentium infection. d , Epithelial reactivity gene expression in C. rodentium infected Rag1 -/- . Ret Δ mice and their Rag1 -/- . Ret fl littermate controls. Rag1 -/- . Ret fl n=15; Rag1 -/- . Ret Δ n=17. e , Weight loss in C. rodentium infected Rag1 -/- . Ret Δ mice and their Rag1 -/- . Ret fl littermate controls. Rag1 -/- . Ret fl n=8; Rag1 -/- . Ret Δ n=8. f , Survival curves in C. rodentium infected Rag1 -/- . Ret Δ mice and their Rag1 -/- . Ret fl littermate controls. Rag1 -/- . Ret fl n=8; Rag1 -/- . Ret Δ n=8. g , C. rodentium translocation to the liver of Ret Δ and their Ret fl littermate controls at day 6 post-infection. n=6. h , MacConkey plates of liver cell suspensions from Ret Δ and their Ret fl littermate controls at day 6 after C. rodentium infection. i , Whole-body imaging of Ret Δ and their Ret fl littermate controls at day 6 after luciferase-expressing C. rodentium infection. j , C. rodentium infection burden. Ret fl n=8; Ret Δ n=8. k , Innate IL-22 in in C. rodentium infected Ret Δ mice and their Ret fl littermate controls. Ret fl n=8; Ret Δ n=8. Data are representative of 3-4 independent experiments. Error bars show s.e.m. ns not significant. Error bars show s.e.m. *P

    Techniques Used: Infection, Mouse Assay, Translocation Assay, Imaging, Luciferase, Expressing

    The neurotrophic factor receptor RET drives enteric ILC3-derived IL-22. a , LTi, NCR - and NCR + ILC3 subsets, T cells (T), B cells (B), Dendritic cells (Dc), Macrophages (Mø), enteric Neurons (N) and mucosal Glial cells (G). b , Ret GFP ILC3. c , Left: Ret GFP gut. White: GFP. Right: ILC3 aggregates. d , Cryptopatches (CP), immature (iILF) and mature (mILF) isolated lymphoid follicles. Green: RET/GFP; Blue: RORγt; Red: B220. e , Ret GFP chimeras. n=15. f,g , Ret GFP chimeras. Ret WT/GFP n=25; Ret GFP/GFP n=22. h , Ret MEN2B mice. n=7. Scale bars: 1mm (c left, e); 50µm (c right); 30µm (d). Data are representative of 4 independent experiments. Error bars show s.e.m. *P
    Figure Legend Snippet: The neurotrophic factor receptor RET drives enteric ILC3-derived IL-22. a , LTi, NCR - and NCR + ILC3 subsets, T cells (T), B cells (B), Dendritic cells (Dc), Macrophages (Mø), enteric Neurons (N) and mucosal Glial cells (G). b , Ret GFP ILC3. c , Left: Ret GFP gut. White: GFP. Right: ILC3 aggregates. d , Cryptopatches (CP), immature (iILF) and mature (mILF) isolated lymphoid follicles. Green: RET/GFP; Blue: RORγt; Red: B220. e , Ret GFP chimeras. n=15. f,g , Ret GFP chimeras. Ret WT/GFP n=25; Ret GFP/GFP n=22. h , Ret MEN2B mice. n=7. Scale bars: 1mm (c left, e); 50µm (c right); 30µm (d). Data are representative of 4 independent experiments. Error bars show s.e.m. *P

    Techniques Used: Derivative Assay, Isolation, Mouse Assay

    Enteric inflammation in mice with altered RET signals. Mice were treated with DSS in the drinking water. a , Weight loss of DSS treated Ret Δ mice and their littermate controls . Ret fl n=8; Ret Δ n=8. b , T cell derived IL-22 in Ret Δ mice and their WT littermate controls after DSS treatment. Ret fl n=8; Ret Δ n=8. c , Weight loss of DSS treated Ret MEN2B mice and their WT littermate controls. Ret WT n=8; Ret MEN2B n=8. d , T cell derived IL-22 in Ret MEN2B mice and their WT littermate controls. Ret WT n=8; Ret MEN2B n=8. e , Intestinal villi and crypt morphology. Ret fl n=6; Ret Δ n=6. f , Epithelial reactivity gene expression in DSS treated Ret Δ mice in comparison to their WT littermate controls. n=8. g , Tissue repair gene expression in DSS treated Ret Δ mice in comparison to their WT littermate controls. n=4. Data are representative of 3-4 independent experiments. Error bars show s.e.m. ns not significant. Error bars show s.e.m. *P
    Figure Legend Snippet: Enteric inflammation in mice with altered RET signals. Mice were treated with DSS in the drinking water. a , Weight loss of DSS treated Ret Δ mice and their littermate controls . Ret fl n=8; Ret Δ n=8. b , T cell derived IL-22 in Ret Δ mice and their WT littermate controls after DSS treatment. Ret fl n=8; Ret Δ n=8. c , Weight loss of DSS treated Ret MEN2B mice and their WT littermate controls. Ret WT n=8; Ret MEN2B n=8. d , T cell derived IL-22 in Ret MEN2B mice and their WT littermate controls. Ret WT n=8; Ret MEN2B n=8. e , Intestinal villi and crypt morphology. Ret fl n=6; Ret Δ n=6. f , Epithelial reactivity gene expression in DSS treated Ret Δ mice in comparison to their WT littermate controls. n=8. g , Tissue repair gene expression in DSS treated Ret Δ mice in comparison to their WT littermate controls. n=4. Data are representative of 3-4 independent experiments. Error bars show s.e.m. ns not significant. Error bars show s.e.m. *P

    Techniques Used: Mouse Assay, Derivative Assay, Expressing

    4) Product Images from "A Subset of CCL25-Induced Gut-Homing T Cells Affects Intestinal Immunity to Infection and Cancer"

    Article Title: A Subset of CCL25-Induced Gut-Homing T Cells Affects Intestinal Immunity to Infection and Cancer

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00271

    Subcutaneous co-delivery of antigen and CCL25 induces the development of α 4 β 7 high Th1 T cells. (A) Dylight 488-labeled CCL25 (0.06 mg/kg) was subcutaneously injected into mice. Two hours later, the dLNs were harvested. Tissue sections from dLN were stained with hamster anti-mouse CD11c antibody (clone N418, BioLegend), or rat anti-mouse CD31 Antibody (MEC13.3, BioLegend) overnight at 4°C. Following three washes in PBS, the sections were incubated with the secondary antibody Alexa Fluor® 546 goat anti-hamster IgG or Alexa Fluor® 555 goat anti-rat IgG (Life Technologies) for 30 min at room temperature followed by three washes. Sections were mounted on microscopy slides with 4′,6-diamidino-2-phenylindole (DAPI) mounting medium (Vectashield). Images taken by wide field fluorescence microscopy are shown. Scale bar, 20 μM. (B–D) Female Marilyn- rag2 −/− mice were immunized by subcutaneous administration of 5 × 10 6 male-derived splenocytes in saline solution or the presence of 0.06 mg/kg CCL25. One week later T cells were separately harvested from axillary (draining LNs, dLN), PP, mesenteric LN (non-draining LN, ndLN), and spleen. Marilyn (MY) T cells were identified by gating on the CD4 + Vβ6 + T cell population. The number of α 4 β 7 high (B) , IFN-γ- (C) or IL-17-expressing (D) T cells was measured by flow cytometry. The mean number of T cells from two independent experiments of identical design is shown below each set of dot-plots (±SD, n = 3). (E–J) OT-II naïve T cells from (10 7 /mouse) were labeled with CFSE (4 μM) and injected intravenously into syngeneic recipients, which were immunized 3 h later by s.c. administration of 0.5 μg OVA-DEC plus 50 μg poly IC adjuvant (InvivoGen) re-suspended in saline solution or in the presence of CCL25 (0.06 mg/kg). T cells were separately harvested from draining LN (axillary, dLN), mesenteric LNs (mLNs), Peyer's Patches (PPs) and spleen 3 (E–G) and 5 days (H–J) later. Expression of α 4 β 7 (E,H) and IFN-γ (F,I) or IL-17 (G,J) by CFSE low T cells was assessed by flow cytometry by gating on CD4 + Vα2 + T cells (OTII TCR). Staining with an isotype-matched control antibody in draining lymph nodes of mice injected with adjuvant alone are shown on top of each set of dot plots. The mean values obtained in at least 3 experiments of identical design are shown below each set of representative dot plots (±SD). * p
    Figure Legend Snippet: Subcutaneous co-delivery of antigen and CCL25 induces the development of α 4 β 7 high Th1 T cells. (A) Dylight 488-labeled CCL25 (0.06 mg/kg) was subcutaneously injected into mice. Two hours later, the dLNs were harvested. Tissue sections from dLN were stained with hamster anti-mouse CD11c antibody (clone N418, BioLegend), or rat anti-mouse CD31 Antibody (MEC13.3, BioLegend) overnight at 4°C. Following three washes in PBS, the sections were incubated with the secondary antibody Alexa Fluor® 546 goat anti-hamster IgG or Alexa Fluor® 555 goat anti-rat IgG (Life Technologies) for 30 min at room temperature followed by three washes. Sections were mounted on microscopy slides with 4′,6-diamidino-2-phenylindole (DAPI) mounting medium (Vectashield). Images taken by wide field fluorescence microscopy are shown. Scale bar, 20 μM. (B–D) Female Marilyn- rag2 −/− mice were immunized by subcutaneous administration of 5 × 10 6 male-derived splenocytes in saline solution or the presence of 0.06 mg/kg CCL25. One week later T cells were separately harvested from axillary (draining LNs, dLN), PP, mesenteric LN (non-draining LN, ndLN), and spleen. Marilyn (MY) T cells were identified by gating on the CD4 + Vβ6 + T cell population. The number of α 4 β 7 high (B) , IFN-γ- (C) or IL-17-expressing (D) T cells was measured by flow cytometry. The mean number of T cells from two independent experiments of identical design is shown below each set of dot-plots (±SD, n = 3). (E–J) OT-II naïve T cells from (10 7 /mouse) were labeled with CFSE (4 μM) and injected intravenously into syngeneic recipients, which were immunized 3 h later by s.c. administration of 0.5 μg OVA-DEC plus 50 μg poly IC adjuvant (InvivoGen) re-suspended in saline solution or in the presence of CCL25 (0.06 mg/kg). T cells were separately harvested from draining LN (axillary, dLN), mesenteric LNs (mLNs), Peyer's Patches (PPs) and spleen 3 (E–G) and 5 days (H–J) later. Expression of α 4 β 7 (E,H) and IFN-γ (F,I) or IL-17 (G,J) by CFSE low T cells was assessed by flow cytometry by gating on CD4 + Vα2 + T cells (OTII TCR). Staining with an isotype-matched control antibody in draining lymph nodes of mice injected with adjuvant alone are shown on top of each set of dot plots. The mean values obtained in at least 3 experiments of identical design are shown below each set of representative dot plots (±SD). * p

    Techniques Used: Labeling, Injection, Mouse Assay, Staining, Incubation, Microscopy, Fluorescence, Derivative Assay, Expressing, Flow Cytometry, Cytometry

    5) Product Images from "Combined immune checkpoint blockade as a therapeutic strategy for BRCA1-mutated breast cancer"

    Article Title: Combined immune checkpoint blockade as a therapeutic strategy for BRCA1-mutated breast cancer

    Journal: Science translational medicine

    doi: 10.1126/scitranslmed.aal4922

    Combination therapy with checkpoint inhibitors curtails the growth of Brca1 -deficient tumors Graph depicting the percentages of PD-L1 + ( A ) tumor cells and ( B ) stromal cells (Lin + ) within mammary tumors harvested from MMTV-cre/Brca1 fl/fl /p53 +/ − , MMTV-Neu, MMTV-PyMT, MMTV-Wnt1 , and p53 +/ − mice. PD-L1 expression was determined by flow cytometry on freshly harvested tumors, and the percentage of positive cells was determined by comparing PD-L1 expression to an isotype-matched control antibody. Data are means ± SEM; each data point depicts an individual tumor. * P
    Figure Legend Snippet: Combination therapy with checkpoint inhibitors curtails the growth of Brca1 -deficient tumors Graph depicting the percentages of PD-L1 + ( A ) tumor cells and ( B ) stromal cells (Lin + ) within mammary tumors harvested from MMTV-cre/Brca1 fl/fl /p53 +/ − , MMTV-Neu, MMTV-PyMT, MMTV-Wnt1 , and p53 +/ − mice. PD-L1 expression was determined by flow cytometry on freshly harvested tumors, and the percentage of positive cells was determined by comparing PD-L1 expression to an isotype-matched control antibody. Data are means ± SEM; each data point depicts an individual tumor. * P

    Techniques Used: Mouse Assay, Expressing, Flow Cytometry, Cytometry

    BRCA1 -mutated TNBCs exhibit prominent lymphocytic infiltrate and PD-L1 expression ( A ) Representative H E image of a BRCA1 -mutated TNBC. Scale bar, 100 μm. ( B ) Analysis of matched BRCA1 -positive TNBC patient stromal TIL populations for H E, OPAL staining, stromal, and intratumoral PD-L1 expression ( n = 16). ( C ) BRCA1 -mutated TNBC. H E and accompanying section immunostained for PD-L1. Scale bars, 100 m m. ( D ) Representative BRCA1 -mutated TNBC patient sample with high stromal TILs. The inset indicates an area with a high number of CD3 + (*), CD4 + (**), and CD8 + . Scale bars, 100 m m (main image) and 20 μm (inset). ( E ) BRCA1 -mutated TNBCs. The OPAL staining panel includes tumor marker CK18 (yellow), CD3 (red), CD4 (white), CD8 (green), FOXP3 (orange), PD-L1 (cyan), and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 100 μm. ( F ) OPAL staining for PD-L1 and CK18, revealing high intratumoral PD-L1 expression. Scale bar, 100 μm. ( G ) Sankey plot of a fluorescence-activated cell sorting (FACS) profile of CD3 + TILs from a BRCA1 -mutated tumor, showing percentages of CD8 + and CD4 + ).
    Figure Legend Snippet: BRCA1 -mutated TNBCs exhibit prominent lymphocytic infiltrate and PD-L1 expression ( A ) Representative H E image of a BRCA1 -mutated TNBC. Scale bar, 100 μm. ( B ) Analysis of matched BRCA1 -positive TNBC patient stromal TIL populations for H E, OPAL staining, stromal, and intratumoral PD-L1 expression ( n = 16). ( C ) BRCA1 -mutated TNBC. H E and accompanying section immunostained for PD-L1. Scale bars, 100 m m. ( D ) Representative BRCA1 -mutated TNBC patient sample with high stromal TILs. The inset indicates an area with a high number of CD3 + (*), CD4 + (**), and CD8 + . Scale bars, 100 m m (main image) and 20 μm (inset). ( E ) BRCA1 -mutated TNBCs. The OPAL staining panel includes tumor marker CK18 (yellow), CD3 (red), CD4 (white), CD8 (green), FOXP3 (orange), PD-L1 (cyan), and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 100 μm. ( F ) OPAL staining for PD-L1 and CK18, revealing high intratumoral PD-L1 expression. Scale bar, 100 μm. ( G ) Sankey plot of a fluorescence-activated cell sorting (FACS) profile of CD3 + TILs from a BRCA1 -mutated tumor, showing percentages of CD8 + and CD4 + ).

    Techniques Used: Expressing, Staining, Marker, Fluorescence, FACS

    6) Product Images from "Positron emission tomography imaging of tumor angiogenesis and monitoring of antiangiogenic efficacy using the novel tetrameric peptide probe 64Cu-cyclam-RAFT-c(-RGDfK-)4"

    Article Title: Positron emission tomography imaging of tumor angiogenesis and monitoring of antiangiogenic efficacy using the novel tetrameric peptide probe 64Cu-cyclam-RAFT-c(-RGDfK-)4

    Journal: Angiogenesis

    doi: 10.1007/s10456-012-9281-1

    Transverse and coronal PET images of s.c. HuH-7 tumor-bearing mice at 3 h after i.v. injection of 64 Cu-cyclam-RAFT-c(-RGDfK-) 4 (11.1 MBq) on the day after daily i.p. injections of ( a ) vehicle alone (50 μl of DMSO) or ( b ) TSU-68 (75 mg kg −1 d −1 in 50 μl of DMSO) for 14 days (n = 4 mice for each group). The arrows indicate the tumor location. Representative autoradiographic examination ( c , e ) and CD31 immunofluorescence staining ( d , f ) with the same whole-tumor sections from ( c , d ) vehicle- and ( e , f ) TSU-68-treated tumors excised after PET imaging. g MVD, ha′ SUV mean , and hb′ SUV max were compared between TSU-68- and vehicle-treated tumors. All data presented in a – h are from the same set of experimental groups
    Figure Legend Snippet: Transverse and coronal PET images of s.c. HuH-7 tumor-bearing mice at 3 h after i.v. injection of 64 Cu-cyclam-RAFT-c(-RGDfK-) 4 (11.1 MBq) on the day after daily i.p. injections of ( a ) vehicle alone (50 μl of DMSO) or ( b ) TSU-68 (75 mg kg −1 d −1 in 50 μl of DMSO) for 14 days (n = 4 mice for each group). The arrows indicate the tumor location. Representative autoradiographic examination ( c , e ) and CD31 immunofluorescence staining ( d , f ) with the same whole-tumor sections from ( c , d ) vehicle- and ( e , f ) TSU-68-treated tumors excised after PET imaging. g MVD, ha′ SUV mean , and hb′ SUV max were compared between TSU-68- and vehicle-treated tumors. All data presented in a – h are from the same set of experimental groups

    Techniques Used: Positron Emission Tomography, Mouse Assay, Injection, Immunofluorescence, Staining, Imaging

    a Tumor growth curve of s.c. HuH-7 tumor-bearing mice treated with daily i.p. injections of TSU-68 (75 mg kg −1 d −1 in 50 μl of DMSO) or the vehicle alone (50 μl of DMSO). Data are presented as the mean ± SD of tumor volumes. Day 0: the day before treatment; days 1–14: treatment days; day 15: 1 day after treatment. b Changes in the body weight of TSU-68- and vehicle-treated mice. c Image of TSU-68- and vehicle-treated tumors excised on day 15. d Representative immunofluorescence staining of tumor sections from TSU-68- and vehicle-treated tumors using anti-mouse CD31 antibody ( green ). Scale bar = 1,000 μm. e The tumor MVD presented as the percentage of the CD31-positive area was compared between tumors from TSU-68- and vehicle-treated mice. All data presented in a – e are from the same set of experimental groups (n = 6 for each group)
    Figure Legend Snippet: a Tumor growth curve of s.c. HuH-7 tumor-bearing mice treated with daily i.p. injections of TSU-68 (75 mg kg −1 d −1 in 50 μl of DMSO) or the vehicle alone (50 μl of DMSO). Data are presented as the mean ± SD of tumor volumes. Day 0: the day before treatment; days 1–14: treatment days; day 15: 1 day after treatment. b Changes in the body weight of TSU-68- and vehicle-treated mice. c Image of TSU-68- and vehicle-treated tumors excised on day 15. d Representative immunofluorescence staining of tumor sections from TSU-68- and vehicle-treated tumors using anti-mouse CD31 antibody ( green ). Scale bar = 1,000 μm. e The tumor MVD presented as the percentage of the CD31-positive area was compared between tumors from TSU-68- and vehicle-treated mice. All data presented in a – e are from the same set of experimental groups (n = 6 for each group)

    Techniques Used: Mouse Assay, Immunofluorescence, Staining

    a Flow cytometric analysis for detecting α V β 3 integrin expression on human hepatocellular carcinoma HuH-7 cells in culture. HEK293(β 1 ) and HEK293(β 3 ) were used as the negative and positive controls, respectively. b Immunohistochemical staining of human α V β 3 integrin in HuH-7, HEK293(β 1 ), and HEK293(β 3 ) tumor xenografts. c HE staining and CD31 immunohistochemical staining of tumor vasculature in HuH-7 tumor xenografts
    Figure Legend Snippet: a Flow cytometric analysis for detecting α V β 3 integrin expression on human hepatocellular carcinoma HuH-7 cells in culture. HEK293(β 1 ) and HEK293(β 3 ) were used as the negative and positive controls, respectively. b Immunohistochemical staining of human α V β 3 integrin in HuH-7, HEK293(β 1 ), and HEK293(β 3 ) tumor xenografts. c HE staining and CD31 immunohistochemical staining of tumor vasculature in HuH-7 tumor xenografts

    Techniques Used: Flow Cytometry, Expressing, Immunohistochemistry, Staining

    7) Product Images from "The Phosphatase PTP-PEST/PTPN12 Regulates Endothelial Cell Migration and Adhesion, but Not Permeability, and Controls Vascular Development and Embryonic Viability *"

    Article Title: The Phosphatase PTP-PEST/PTPN12 Regulates Endothelial Cell Migration and Adhesion, but Not Permeability, and Controls Vascular Development and Embryonic Viability *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.387456

    Generation and characterization of PTP-PEST-deficient primary endothelial cells. A , protocol used to generate PTP-PEST-deficient endothelial cells. Adult mice were fed for 5 consecutive days ( D1–5 ) with tamoxifen ( TAM ). On day 15, lung cells were harvested and seeded in culture. Two purifications of endothelial cells were done on days 21 and 25 using anti-CD102 antibody-coupled magnetic beads. After an expansion on day 28, cells were used for experimentation on day 31. B , expression of PTP-PEST and Csk (as control) in primary endothelial cells from tamoxifen-fed Ptpn12 +/+ ; UBC-Cre-ERT2 + (control ( CTRL )) or tamoxifen-fed Ptpn12 fl/fl ; UBC-Cre-ERT2 + (knock-out ( KO )) mice was analyzed by immunoblotting. Data are representative of > 10 experiments. C , flow cytometry analyses of surface markers on primary endothelial cells from control ( blue ) and KO ( red ) mice. Staining with an isotype control antibody is depicted by the filled gray curves . Data are representative of at least two experiments. D , primary endothelial cells from control and KO mice were seeded in growth medium containing endothelial growth supplement. They were then harvested at different times (in hours) and counted. Error bars representing standard deviations of values from duplicate wells are shown. Data are representative of two experiments.
    Figure Legend Snippet: Generation and characterization of PTP-PEST-deficient primary endothelial cells. A , protocol used to generate PTP-PEST-deficient endothelial cells. Adult mice were fed for 5 consecutive days ( D1–5 ) with tamoxifen ( TAM ). On day 15, lung cells were harvested and seeded in culture. Two purifications of endothelial cells were done on days 21 and 25 using anti-CD102 antibody-coupled magnetic beads. After an expansion on day 28, cells were used for experimentation on day 31. B , expression of PTP-PEST and Csk (as control) in primary endothelial cells from tamoxifen-fed Ptpn12 +/+ ; UBC-Cre-ERT2 + (control ( CTRL )) or tamoxifen-fed Ptpn12 fl/fl ; UBC-Cre-ERT2 + (knock-out ( KO )) mice was analyzed by immunoblotting. Data are representative of > 10 experiments. C , flow cytometry analyses of surface markers on primary endothelial cells from control ( blue ) and KO ( red ) mice. Staining with an isotype control antibody is depicted by the filled gray curves . Data are representative of at least two experiments. D , primary endothelial cells from control and KO mice were seeded in growth medium containing endothelial growth supplement. They were then harvested at different times (in hours) and counted. Error bars representing standard deviations of values from duplicate wells are shown. Data are representative of two experiments.

    Techniques Used: Mouse Assay, Magnetic Beads, Expressing, Knock-Out, Flow Cytometry, Cytometry, Staining

    Analyses of Ptpn12 fl/fl ; Tie2-Cre + embryos. A , E9.5 and E10.5 embryos from Ptpn12 fl/fl ; Tie2-Cre + ( KO ) and Ptpn12 +/+ ; Tie2-Cre + (control ( CTRL )) mice were photographed. Data are representative of > 9 experiments. B , whole-mount staining with anti-CD31 antibodies of E9.5 ( panels a–i ) and E10.5 ( panels j–r ) embryos from KO and control mice. Embryos from a constitutive PTP-PEST-deficient mouse ( TOTAL KO ) are also shown for comparison. Regions defined in the blue-bordered insets in panels a–c and j–l were analyzed at greater magnification in panels d–i and m–r. Scale bars for panels A–C represent 800 μm, whereas those for panels J–L represent 1000 μm. Data are representative of two (E9.5) or three (E10.5) experiments. C , toluidine blue-stained cross-sections of E10.5 embryos from control and KO mice. For each mouse, the region defined in the inset in the left-hand panel was analyzed at greater magnification in the right-hand panel. Arrows show the location of the dorsal aorta. D , flow cytometry analyses of cells from disaggregated E10.5 embryos from control and KO mice. Analyses were gated on EYFP-positive cells. The percentages of cells positive for the various markers are shown in the upper right quadrant. Data are representative of three experiments.
    Figure Legend Snippet: Analyses of Ptpn12 fl/fl ; Tie2-Cre + embryos. A , E9.5 and E10.5 embryos from Ptpn12 fl/fl ; Tie2-Cre + ( KO ) and Ptpn12 +/+ ; Tie2-Cre + (control ( CTRL )) mice were photographed. Data are representative of > 9 experiments. B , whole-mount staining with anti-CD31 antibodies of E9.5 ( panels a–i ) and E10.5 ( panels j–r ) embryos from KO and control mice. Embryos from a constitutive PTP-PEST-deficient mouse ( TOTAL KO ) are also shown for comparison. Regions defined in the blue-bordered insets in panels a–c and j–l were analyzed at greater magnification in panels d–i and m–r. Scale bars for panels A–C represent 800 μm, whereas those for panels J–L represent 1000 μm. Data are representative of two (E9.5) or three (E10.5) experiments. C , toluidine blue-stained cross-sections of E10.5 embryos from control and KO mice. For each mouse, the region defined in the inset in the left-hand panel was analyzed at greater magnification in the right-hand panel. Arrows show the location of the dorsal aorta. D , flow cytometry analyses of cells from disaggregated E10.5 embryos from control and KO mice. Analyses were gated on EYFP-positive cells. The percentages of cells positive for the various markers are shown in the upper right quadrant. Data are representative of three experiments.

    Techniques Used: Mouse Assay, Staining, Flow Cytometry, Cytometry

    8) Product Images from "The Choroid Plexus Functions as a Niche for T-Cell Stimulation Within the Central Nervous System"

    Article Title: The Choroid Plexus Functions as a Niche for T-Cell Stimulation Within the Central Nervous System

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01066

    Intracerebroventricularly (ICV)-injected, resting ovalbumin (OVA)-specific CD4 T cells undergo proliferation within the choroid plexus (CP) in a cerebrospinal fluid-antigen-dependent manner. Interferon gamma was injected intra-cisterna magna to wild-type mice, either alone or together with OVA 323–339 or MOG 35–55 (as a control peptide). After 24 h, the mice were ICV-injected with carboxyfluorescein succinimidyl ester (CFSE)-labeled, resting OVA-specific Th1 cells, either without a peptide (Control; n = 5), with OVA 323–339 (OVA; n = 7), or with MOG 35–55 [myelin oligodendrocyte glycoprotein (MOG); n = 4]. At 3-day post-injection, the lateral ventricle (LV) CPs of these mice were analyzed by flow cytometry and immunohistochemistry (IHC). (A) Flow cytometry of isolated CPs. The cellular fraction gated on mononuclear cells shows CD45 + CD4 + and CFSE + T cells. (B–F) IHC images of LV CPs obtained from the control and from the OVA-injected mice. (B) Representative brain sections of OVA-injected and control mice, immunolabeled with anti-intercellular adhesion molecule 1 (ICAM-1) (green). The graph shows fold change in ICAM-1 expression in the LV CPs of OVA-injected mice, normalized to the control mice. Scale bars represent 50 µm. (C) Representative brain sections of OVA-injected and control mice, immunolabeled with anti-CD4 (green), anti-vascular cell adhesion molecule 1 (VCAM-1) (blue), and a DAPI counterstained (gray). Scale bars represent 200 µm (top left and middle left panels), 50 µm (top right and middle right panels), and 10 µm (bottom left panel). The bottom right panel shows a 3D reconstruction of z-sections (25.9 µm overall, 0.7 µm/slice) of the framed area. (D–F) Analyses of the interactions between T cells and myeloid cells, and the proliferation of T cells within the CP. (D) Representative brain sections of OVA-injected mice, immunolabeled with anti-CD4 (green), anti-Iba-1 (red), and a TO-PRO-3 nuclear counterstain (blue). The yellow arrows indicate co-localization of CD4 and Iba-1. The right panel shows a 3D reconstruction of z-sections (9.5 µm overall, 0.5 µm/slice) of the framed area. Scale bars represent 50 µm (left) and 10 µm (middle). (E) Representative brain sections of OVA-injected mice immunolabeled with anti-Ki-67 (red) and anti-CD4 (blue). The yellow arrows indicate proliferating CD4 T cells in the CP. The right panel shows a 3D reconstruction of z-sections (22 µm overall, 2 µm/slice) of the framed area. Scale bars represent 50 µm (left) and 10 µm (right). (F) Expression pattern of CFSE and Ki-67 in CD4 + T cells, which were detected in three 40-µm brain sections of a single LV CP from a mouse injected with OVA and with OVA-specific T cells. (A,B) Each symbol represents one LV CP from an individual mouse. Bars represent means ± SEM. * P
    Figure Legend Snippet: Intracerebroventricularly (ICV)-injected, resting ovalbumin (OVA)-specific CD4 T cells undergo proliferation within the choroid plexus (CP) in a cerebrospinal fluid-antigen-dependent manner. Interferon gamma was injected intra-cisterna magna to wild-type mice, either alone or together with OVA 323–339 or MOG 35–55 (as a control peptide). After 24 h, the mice were ICV-injected with carboxyfluorescein succinimidyl ester (CFSE)-labeled, resting OVA-specific Th1 cells, either without a peptide (Control; n = 5), with OVA 323–339 (OVA; n = 7), or with MOG 35–55 [myelin oligodendrocyte glycoprotein (MOG); n = 4]. At 3-day post-injection, the lateral ventricle (LV) CPs of these mice were analyzed by flow cytometry and immunohistochemistry (IHC). (A) Flow cytometry of isolated CPs. The cellular fraction gated on mononuclear cells shows CD45 + CD4 + and CFSE + T cells. (B–F) IHC images of LV CPs obtained from the control and from the OVA-injected mice. (B) Representative brain sections of OVA-injected and control mice, immunolabeled with anti-intercellular adhesion molecule 1 (ICAM-1) (green). The graph shows fold change in ICAM-1 expression in the LV CPs of OVA-injected mice, normalized to the control mice. Scale bars represent 50 µm. (C) Representative brain sections of OVA-injected and control mice, immunolabeled with anti-CD4 (green), anti-vascular cell adhesion molecule 1 (VCAM-1) (blue), and a DAPI counterstained (gray). Scale bars represent 200 µm (top left and middle left panels), 50 µm (top right and middle right panels), and 10 µm (bottom left panel). The bottom right panel shows a 3D reconstruction of z-sections (25.9 µm overall, 0.7 µm/slice) of the framed area. (D–F) Analyses of the interactions between T cells and myeloid cells, and the proliferation of T cells within the CP. (D) Representative brain sections of OVA-injected mice, immunolabeled with anti-CD4 (green), anti-Iba-1 (red), and a TO-PRO-3 nuclear counterstain (blue). The yellow arrows indicate co-localization of CD4 and Iba-1. The right panel shows a 3D reconstruction of z-sections (9.5 µm overall, 0.5 µm/slice) of the framed area. Scale bars represent 50 µm (left) and 10 µm (middle). (E) Representative brain sections of OVA-injected mice immunolabeled with anti-Ki-67 (red) and anti-CD4 (blue). The yellow arrows indicate proliferating CD4 T cells in the CP. The right panel shows a 3D reconstruction of z-sections (22 µm overall, 2 µm/slice) of the framed area. Scale bars represent 50 µm (left) and 10 µm (right). (F) Expression pattern of CFSE and Ki-67 in CD4 + T cells, which were detected in three 40-µm brain sections of a single LV CP from a mouse injected with OVA and with OVA-specific T cells. (A,B) Each symbol represents one LV CP from an individual mouse. Bars represent means ± SEM. * P

    Techniques Used: Injection, Mouse Assay, Labeling, Flow Cytometry, Cytometry, Immunohistochemistry, Isolation, Immunolabeling, Expressing

    9) Product Images from "cAMP Response Element Binding Protein Is Required for Differentiation of Respiratory Epithelium during Murine Development"

    Article Title: cAMP Response Element Binding Protein Is Required for Differentiation of Respiratory Epithelium during Murine Development

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017843

    Morphological analysis of lung development in lungs of Creb1 −/− mice. Haematoxylin and eosin-stained tissue sections from E15.5–E18.5 Creb1 −/− and wildtype lungs (A–H). Lung morphology of Creb1 −/− and wildtype fetal mice was similar at E15.5 (A, B) and E16.5 (C, D). At E17.5 proximal and distal airways of Creb1 −/− mice failed to expand and showed compacted tissue morphology (F). Lungs of E18.5 Creb1 −/− mice showed a comparable morphology to E17.5 Creb1 −/− lungs (H). In comparison littermate controls at E17.5–E18.5 showed normal airway expansion (E, G). Lungs of E17.5 Creb1 −/− mice were smaller than wildtype, though in approximate proportion to a reduced body size of Creb1 −/− mice (I). Immunohistochemical analysis for the cell division marker Ki67 in lungs of E18.5 wildtype (J) and Creb1 −/− (K) mice. Quantification of Ki67-positive and -negative cells ( n = 3) in total lung showed a small increase in cell proliferation in lungs of Creb1 −/− mice at E18.5 (L). TUNEL analysis for apoptotic nuclei showed very rare, but comparable numbers of apoptotic cells in E18.5 wildtype (M) and Creb1 −/− (N) lungs (arrows indicate apoptotic nuclei). Error bars represent SEM. Asterisk (*) indicates p
    Figure Legend Snippet: Morphological analysis of lung development in lungs of Creb1 −/− mice. Haematoxylin and eosin-stained tissue sections from E15.5–E18.5 Creb1 −/− and wildtype lungs (A–H). Lung morphology of Creb1 −/− and wildtype fetal mice was similar at E15.5 (A, B) and E16.5 (C, D). At E17.5 proximal and distal airways of Creb1 −/− mice failed to expand and showed compacted tissue morphology (F). Lungs of E18.5 Creb1 −/− mice showed a comparable morphology to E17.5 Creb1 −/− lungs (H). In comparison littermate controls at E17.5–E18.5 showed normal airway expansion (E, G). Lungs of E17.5 Creb1 −/− mice were smaller than wildtype, though in approximate proportion to a reduced body size of Creb1 −/− mice (I). Immunohistochemical analysis for the cell division marker Ki67 in lungs of E18.5 wildtype (J) and Creb1 −/− (K) mice. Quantification of Ki67-positive and -negative cells ( n = 3) in total lung showed a small increase in cell proliferation in lungs of Creb1 −/− mice at E18.5 (L). TUNEL analysis for apoptotic nuclei showed very rare, but comparable numbers of apoptotic cells in E18.5 wildtype (M) and Creb1 −/− (N) lungs (arrows indicate apoptotic nuclei). Error bars represent SEM. Asterisk (*) indicates p

    Techniques Used: Mouse Assay, Staining, Immunohistochemistry, Marker, TUNEL Assay

    10) Product Images from "Immune‐mediated ECM depletion improves tumour perfusion and payload delivery"

    Article Title: Immune‐mediated ECM depletion improves tumour perfusion and payload delivery

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201910923

    The effects of TNFα‐CSG on tumour ECM and perfusion Comparison of CD8 + T‐cell (red) or ECM content (laminin, red) in tumour (T) and the surrounding normal pancreas (N Pan) for RIP1‐Tag5 mice treated with 5 daily doses on indicated compound. Scale bars: 100 μm. Stiffness analyses of the RIP1‐Tag5 tumours following treatment with 4 × daily i.v. injections of indicated compounds. Tumour stiffness was analysed on day 5. Left panel: En face quantitative micro‐elastograms showing tumour stiffness (red arrow: stiffest zones). Right panel: The corresponding micrograph of haematoxylin and eosin (HE) staining of the tumours (yellow arrow: ECM‐rich zone). Scale bars: 400 μm. Micrographs show lectin + vessels (green) in 4T1 tumours, after mice were treated with 5 daily injections of indicated compounds and perfused with fluorescein‐labelled lectin to visualise patent blood vessels. Data show average vessel width for individual tumours and mean ± SEM for each group ( n = 4; **P
    Figure Legend Snippet: The effects of TNFα‐CSG on tumour ECM and perfusion Comparison of CD8 + T‐cell (red) or ECM content (laminin, red) in tumour (T) and the surrounding normal pancreas (N Pan) for RIP1‐Tag5 mice treated with 5 daily doses on indicated compound. Scale bars: 100 μm. Stiffness analyses of the RIP1‐Tag5 tumours following treatment with 4 × daily i.v. injections of indicated compounds. Tumour stiffness was analysed on day 5. Left panel: En face quantitative micro‐elastograms showing tumour stiffness (red arrow: stiffest zones). Right panel: The corresponding micrograph of haematoxylin and eosin (HE) staining of the tumours (yellow arrow: ECM‐rich zone). Scale bars: 400 μm. Micrographs show lectin + vessels (green) in 4T1 tumours, after mice were treated with 5 daily injections of indicated compounds and perfused with fluorescein‐labelled lectin to visualise patent blood vessels. Data show average vessel width for individual tumours and mean ± SEM for each group ( n = 4; **P

    Techniques Used: Mouse Assay, Staining

    CD8 + and CD4 + T cells mediate ECM degradation and anti‐tumour effects of TNFα‐CSG BALB/c nude mice bearing 4T1 tumours were treated with 5 daily i.v. injections of TNFα‐CSG in the absence or presence of naïve splenic CD4 + and CD8 + T cells, or both (triple treatment). The cells were injected i.p. on days 10 and 13. On day 16 post‐implantation, the tumours were analysed for weight and volume (B), immune infiltrates (C) and collagen IV content (D). Plots of individual tumour weights (g) and volumes (mm 3 ) and mean ± SEM on day 16 are shown for untreated (UT) and treated groups ( n = 5; * P
    Figure Legend Snippet: CD8 + and CD4 + T cells mediate ECM degradation and anti‐tumour effects of TNFα‐CSG BALB/c nude mice bearing 4T1 tumours were treated with 5 daily i.v. injections of TNFα‐CSG in the absence or presence of naïve splenic CD4 + and CD8 + T cells, or both (triple treatment). The cells were injected i.p. on days 10 and 13. On day 16 post‐implantation, the tumours were analysed for weight and volume (B), immune infiltrates (C) and collagen IV content (D). Plots of individual tumour weights (g) and volumes (mm 3 ) and mean ± SEM on day 16 are shown for untreated (UT) and treated groups ( n = 5; * P

    Techniques Used: Mouse Assay, Injection

    The effects of TNFα‐CSG on immune infiltrates in tumours Co‐staining of RIP1‐Tag5 tumour sections treated with 5 daily doses of indicated compounds. Micrographs show CD8 + T‐cell (red) infiltration relative to collagen IV (A; green) and CD31 + blood vessels (B; green). Scale bars: 100 μm. Left panels show representative flow cytometry plots of quantification of immune cells in 4T1 tumours treated with 5 daily i.v. injections of 0.5 μg of unconjugated TNFα or TNFα‐CSG, or left untreated (UT). Bar charts show mean ± SEM of cell counts in each treatment group (data are shown for one of the two repeated experiments; n = 4; * P
    Figure Legend Snippet: The effects of TNFα‐CSG on immune infiltrates in tumours Co‐staining of RIP1‐Tag5 tumour sections treated with 5 daily doses of indicated compounds. Micrographs show CD8 + T‐cell (red) infiltration relative to collagen IV (A; green) and CD31 + blood vessels (B; green). Scale bars: 100 μm. Left panels show representative flow cytometry plots of quantification of immune cells in 4T1 tumours treated with 5 daily i.v. injections of 0.5 μg of unconjugated TNFα or TNFα‐CSG, or left untreated (UT). Bar charts show mean ± SEM of cell counts in each treatment group (data are shown for one of the two repeated experiments; n = 4; * P

    Techniques Used: Staining, Flow Cytometry, Cytometry

    11) Product Images from "Antibody-Mediated Delivery of VEGFC Ameliorates Experimental Chronic Colitis"

    Article Title: Antibody-Mediated Delivery of VEGFC Ameliorates Experimental Chronic Colitis

    Journal: ACS Pharmacology & Translational Science

    doi: 10.1021/acsptsci.9b00037

    F8-VEGFC induces expansion of the lymphatic vasculature in chronic colitis. (A) Representative pictures of immunofluorescence labeling of the inflamed colon after F8-SIP (upper panels) or F8-VEGFC (lower panels) treatment for LYVE-1 (green), CD31 (red), and Hoechst (blue). Dotted white lines mark the separation between the mucosa (M) and the submucosa (SM). Scale bars: 100 μm. LYVE-1 + LVs were quantified in terms of number (B) and percentage of stained area (C) in 5–8 fields per colon per mouse ( n = 5 per group). CD31+LYVE-1- blood vessels were quantified in terms of number (D) and percentage of stained area (E) in 5–8 fields per colon per mouse ( n = 5 per group). All data are presented as mean ± SD. Statistical significance was determined by two-tailed Student’s t test. Asterisks indicate statistical significance with p
    Figure Legend Snippet: F8-VEGFC induces expansion of the lymphatic vasculature in chronic colitis. (A) Representative pictures of immunofluorescence labeling of the inflamed colon after F8-SIP (upper panels) or F8-VEGFC (lower panels) treatment for LYVE-1 (green), CD31 (red), and Hoechst (blue). Dotted white lines mark the separation between the mucosa (M) and the submucosa (SM). Scale bars: 100 μm. LYVE-1 + LVs were quantified in terms of number (B) and percentage of stained area (C) in 5–8 fields per colon per mouse ( n = 5 per group). CD31+LYVE-1- blood vessels were quantified in terms of number (D) and percentage of stained area (E) in 5–8 fields per colon per mouse ( n = 5 per group). All data are presented as mean ± SD. Statistical significance was determined by two-tailed Student’s t test. Asterisks indicate statistical significance with p

    Techniques Used: Immunofluorescence, Labeling, Staining, Two Tailed Test

    F8-VEGFC reduces macrophage infiltration in chronically inflamed colons. Flow cytometry quantification of total leukocytes pregated on single living cells. (A) Percentage of total CD45+ cells. (B) Representative flow cytometry plots of live cells pregated for CD45 positivity of dendritic cells (DC) and macrophages in inflamed colons of F8-SIP (left panel) and F8-VEGFC (right panel) treated mice. (C) Percentage of F4/80+ macrophages of total CD45+ cells ( n = 8 per group). (D) Percentage of CD11c+F4/80- dendritic cells of total CD45+ ( n = 8 per group). (E) Percentage of CD11c-F480-CD11b+ myeloid cells of total CD45+ cells ( n = 8 per group). (F) Representative pictures of immunofluorescence staining of the inflamed colon after F8-SIP (left panel) or F8-VEGFC (right panel) treatment for F4/80 (green) and Hoechst (blue). Scale bars: 100 μm. (G) Percentage of F4/80-positive staining area in 5–8 fields per colon per mouse ( n = 5 per group).
    Figure Legend Snippet: F8-VEGFC reduces macrophage infiltration in chronically inflamed colons. Flow cytometry quantification of total leukocytes pregated on single living cells. (A) Percentage of total CD45+ cells. (B) Representative flow cytometry plots of live cells pregated for CD45 positivity of dendritic cells (DC) and macrophages in inflamed colons of F8-SIP (left panel) and F8-VEGFC (right panel) treated mice. (C) Percentage of F4/80+ macrophages of total CD45+ cells ( n = 8 per group). (D) Percentage of CD11c+F4/80- dendritic cells of total CD45+ ( n = 8 per group). (E) Percentage of CD11c-F480-CD11b+ myeloid cells of total CD45+ cells ( n = 8 per group). (F) Representative pictures of immunofluorescence staining of the inflamed colon after F8-SIP (left panel) or F8-VEGFC (right panel) treatment for F4/80 (green) and Hoechst (blue). Scale bars: 100 μm. (G) Percentage of F4/80-positive staining area in 5–8 fields per colon per mouse ( n = 5 per group).

    Techniques Used: Flow Cytometry, Mouse Assay, Immunofluorescence, Staining

    EDA-FN is increased in inflamed colon tissue. (A) EDA-FN expression levels were assessed in healthy ( n = 6), ulcerative colitis (UC, n = 24), and Crohn’s disease (CD, n = 19) endoscopic colonic biopsies, and absolute values for the probe 212464_s_at, recognizing EDA-FN, are shown. (B) Representative immunofluorescence pictures of naïve (CTRL, n = 3) and inflamed colons at day 24 post DSS administration (DSS, n = 3) stained for CD31 (red), EDA-FN (green), and Hoechst (blue). Scale bar: 100 μm. All data are presented as mean ± SD. Adjusted p value * p
    Figure Legend Snippet: EDA-FN is increased in inflamed colon tissue. (A) EDA-FN expression levels were assessed in healthy ( n = 6), ulcerative colitis (UC, n = 24), and Crohn’s disease (CD, n = 19) endoscopic colonic biopsies, and absolute values for the probe 212464_s_at, recognizing EDA-FN, are shown. (B) Representative immunofluorescence pictures of naïve (CTRL, n = 3) and inflamed colons at day 24 post DSS administration (DSS, n = 3) stained for CD31 (red), EDA-FN (green), and Hoechst (blue). Scale bar: 100 μm. All data are presented as mean ± SD. Adjusted p value * p

    Techniques Used: Expressing, Immunofluorescence, Staining

    12) Product Images from "Argininosuccinic aciduria fosters neuronal nitrosative stress reversed by Asl gene transfer"

    Article Title: Argininosuccinic aciduria fosters neuronal nitrosative stress reversed by Asl gene transfer

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05972-1

    Neonatal intravenous injection of AAV8.EFS.GFP enables neuronal transduction. a Brain imaged with fluorescence microscope in CD-1 pups injected intravenously with AAV8.EFS. GFP (GFP) and uninjected controls. b , c Representative images of GFP immunostaining in brain at b low and c at higher magnifications in mice injected with the GFP vector and uninjected controls show a decreasing rostro-caudal gradient with preferential transduction of forebrain and midbrain. d Computational quantification of GFP immunostaining showed a significant increase in AAV8.EFS. GFP -injected versus uninjected littermates ( n = 4). e Immunofluorescence of cortical staining for DAPI (blue), GFP (green), and NeuN, GFAP, Olig-2, CD68 (red) identifying neurons, astrocytes, oligodendrocytes and microglial cells, respectively. f Colocalisation measured by Pearson’s coefficient showed a restricted neuronal transduction. Horizontal lines display the mean ± SEM. d Unpaired two-tailed Student’s t test * p
    Figure Legend Snippet: Neonatal intravenous injection of AAV8.EFS.GFP enables neuronal transduction. a Brain imaged with fluorescence microscope in CD-1 pups injected intravenously with AAV8.EFS. GFP (GFP) and uninjected controls. b , c Representative images of GFP immunostaining in brain at b low and c at higher magnifications in mice injected with the GFP vector and uninjected controls show a decreasing rostro-caudal gradient with preferential transduction of forebrain and midbrain. d Computational quantification of GFP immunostaining showed a significant increase in AAV8.EFS. GFP -injected versus uninjected littermates ( n = 4). e Immunofluorescence of cortical staining for DAPI (blue), GFP (green), and NeuN, GFAP, Olig-2, CD68 (red) identifying neurons, astrocytes, oligodendrocytes and microglial cells, respectively. f Colocalisation measured by Pearson’s coefficient showed a restricted neuronal transduction. Horizontal lines display the mean ± SEM. d Unpaired two-tailed Student’s t test * p

    Techniques Used: Injection, Transduction, Fluorescence, Microscopy, Immunostaining, Mouse Assay, Plasmid Preparation, Immunofluorescence, Staining, Two Tailed Test

    13) Product Images from "YAP/TAZ direct commitment and maturation of lymph node fibroblastic reticular cells"

    Article Title: YAP/TAZ direct commitment and maturation of lymph node fibroblastic reticular cells

    Journal: Nature Communications

    doi: 10.1038/s41467-020-14293-1

    YAP/TAZ regulate chemokine expression prior to LTβR engagement. a Immunoblot analyses at indicated time points and comparison of normalized pYAP/YAP ratio at 240 min in cultured FRCs derived from WT mice after stimulation with LTβR agonistic antibody (500 ng/ml) for indicated time points. b Immunoblot analyses of indicated proteins in nuclear (LaminB) and cytoplasmic (GAPDH) fractions of cultured FRCs after treatment with or without LTβR agonistic antibody. c Immunoprecipitation (IP) with anti-IgG or anti-YAP/TAZ (αY/T) antibody in primary cultured FRCs derived immunoblot with indicated antibodies. d Pull-down assay with streptavidin resin in HEK-293T cells after transfection with the streptavidin-binding peptide (SBP)-TAZ4SA, with or without plasmids encoding p52 or RelB and immunoblot analysis with indicated antibodies. e Pull-down assay with streptavidin resin in HEK-293T cells after transfection with the (SBP)-TAZ4SA, with or without plasmids encoding p52 (WT) or p52-Y293A mutants (YA) and immunoblot analysis with indicated antibodies. f Pull-down assay with streptavidin resin in HEK-293T cells after transfection with (SBP)-TAZ4SA or (SBP)-WW domain-deleted TAZ mutant (△WW) with or without plasmids encoding p52 or RelB and immunoblot analysis with indicated antibodies. g Diagram depicting the p52/RelB binding site within the mouse Ccl19 promoter and Ccl19 promoter-driven luciferase constructs containing p52/RelB binding site (WT) or the binding site deletion mutant (Mut). h Comparison of relative luciferase reporter activity using WT and Mut in HEK-293T cells. WT and Mut was co-transfected with or without p52 or p52 mutant (YA) and TAZ or TAZ mutant (△WW) in HEK-293T cells ( n = 8). P values by one-way ANOVA. i Representative images of in situ proximity ligation assay showing localizations of YAP or TAZ and p52 after treatment with or without LTβR agonistic antibody in cultured FRCs. Nuclei are stained with DAPI. Scale bars, 50 µm. j ChIP experiments using IgG or anti-TAZ antibody were performed in MEFs infected with retrovirus encoding CTL or TAZ4SA with or without LTβR agonistic antibody. Unless otherwise denoted, similar findings were observed in three independent experiments. Horizontal bars indicate mean ± SD and P value versus 0 min or Control by two-tailed Student’s t -test. NS, not significant.
    Figure Legend Snippet: YAP/TAZ regulate chemokine expression prior to LTβR engagement. a Immunoblot analyses at indicated time points and comparison of normalized pYAP/YAP ratio at 240 min in cultured FRCs derived from WT mice after stimulation with LTβR agonistic antibody (500 ng/ml) for indicated time points. b Immunoblot analyses of indicated proteins in nuclear (LaminB) and cytoplasmic (GAPDH) fractions of cultured FRCs after treatment with or without LTβR agonistic antibody. c Immunoprecipitation (IP) with anti-IgG or anti-YAP/TAZ (αY/T) antibody in primary cultured FRCs derived immunoblot with indicated antibodies. d Pull-down assay with streptavidin resin in HEK-293T cells after transfection with the streptavidin-binding peptide (SBP)-TAZ4SA, with or without plasmids encoding p52 or RelB and immunoblot analysis with indicated antibodies. e Pull-down assay with streptavidin resin in HEK-293T cells after transfection with the (SBP)-TAZ4SA, with or without plasmids encoding p52 (WT) or p52-Y293A mutants (YA) and immunoblot analysis with indicated antibodies. f Pull-down assay with streptavidin resin in HEK-293T cells after transfection with (SBP)-TAZ4SA or (SBP)-WW domain-deleted TAZ mutant (△WW) with or without plasmids encoding p52 or RelB and immunoblot analysis with indicated antibodies. g Diagram depicting the p52/RelB binding site within the mouse Ccl19 promoter and Ccl19 promoter-driven luciferase constructs containing p52/RelB binding site (WT) or the binding site deletion mutant (Mut). h Comparison of relative luciferase reporter activity using WT and Mut in HEK-293T cells. WT and Mut was co-transfected with or without p52 or p52 mutant (YA) and TAZ or TAZ mutant (△WW) in HEK-293T cells ( n = 8). P values by one-way ANOVA. i Representative images of in situ proximity ligation assay showing localizations of YAP or TAZ and p52 after treatment with or without LTβR agonistic antibody in cultured FRCs. Nuclei are stained with DAPI. Scale bars, 50 µm. j ChIP experiments using IgG or anti-TAZ antibody were performed in MEFs infected with retrovirus encoding CTL or TAZ4SA with or without LTβR agonistic antibody. Unless otherwise denoted, similar findings were observed in three independent experiments. Horizontal bars indicate mean ± SD and P value versus 0 min or Control by two-tailed Student’s t -test. NS, not significant.

    Techniques Used: Expressing, Cell Culture, Derivative Assay, Mouse Assay, Immunoprecipitation, Pull Down Assay, Transfection, Binding Assay, Mutagenesis, Luciferase, Construct, Activity Assay, In Situ, Proximity Ligation Assay, Staining, Chromatin Immunoprecipitation, Infection, CTL Assay, Two Tailed Test

    Depletion of Yap/Taz transforms mesenchymal FRC precursors into adipocytes. a Diagram for analyses of indicated mice at 8-weeks old with or without tamoxifen delivery from 4-weeks old. b , c Representative images and comparisons of perilipin + adipocytes within the inguinal LN (dashed line) in indicated mice ( n = 7). Scale bars, 400 µm. d Representative images of inguinal LN filled with adipocytes in Ltbr-Y / T ∆FRC-YR mice ( n = 6). Right upper panel shows the magnified view of the region within the white dashed box and yellow arrowheads in the right lower panel indicate CCL19-YFP + perilipin + BODIPY + adipocytes. Scale bars, 500 µm (left panel); 100 µm (right lower panel). e Representative images of perilipin + adipocytes along the LYVE-1 + lymphatic vessels (dashed-boxes) in inguinal LN of i- Ltbr-Y / T ∆FRC-YR mice ( n = 6). Scale bar, 400 µm. f , Diagram for primary culture of FRCs derived from i- Ltbr-Y / T ∆FRC-YR mice for 4 days and treatment with EtOH or 4-OHT for their analyses at 2 days after the treatment. g Comparisons of indicated mRNA expression normalized to Gapdh in primary cultured FRCs after treatment with EtOH or 4-OHT for 2 days ( n = 4). h Diagram for adipogenic culture of mesenchymal stem cells (C3H/10T1/2) infected with an adenovirus to induce overexpression of active TAZ (TAZ4SA) for their analyses at 8 days after the infection. i Immunoblot analyses of indicated proteins in mesenchymal stem cells (C3H/10T1/2) infected with an adenovirus to induce overexpression of active TAZ (TAZ4SA) or control. j Representative images of Oil Red O staining in mesenchymal stem cells (C3H/10T1/2) induced with adipogenic cocktail after infected with an adenovirus to induce overexpression of active TAZ (TAZ4SA). k Comparisons of indicated mRNA expression normalized to Gapdh in mesenchymal stem cells (C3H/10T1/2) infected with an adenovirus to induce overexpression of active TAZ (TAZ4SA) for their analyses at 2 days after the infection ( n = 4). l Schematic images proposing the importance of coordination of YAP/TAZ activity and LTβR coupling in FRCs during LN growth and maintenance. Unless otherwise denoted, horizontal bars indicate mean ± SD and P values versus non- Y / T ∆FRC-YR or non-i- Ltbr ∆FRC-YR or EtOH or Control by two‐tailed Mann‐Whitney U test.
    Figure Legend Snippet: Depletion of Yap/Taz transforms mesenchymal FRC precursors into adipocytes. a Diagram for analyses of indicated mice at 8-weeks old with or without tamoxifen delivery from 4-weeks old. b , c Representative images and comparisons of perilipin + adipocytes within the inguinal LN (dashed line) in indicated mice ( n = 7). Scale bars, 400 µm. d Representative images of inguinal LN filled with adipocytes in Ltbr-Y / T ∆FRC-YR mice ( n = 6). Right upper panel shows the magnified view of the region within the white dashed box and yellow arrowheads in the right lower panel indicate CCL19-YFP + perilipin + BODIPY + adipocytes. Scale bars, 500 µm (left panel); 100 µm (right lower panel). e Representative images of perilipin + adipocytes along the LYVE-1 + lymphatic vessels (dashed-boxes) in inguinal LN of i- Ltbr-Y / T ∆FRC-YR mice ( n = 6). Scale bar, 400 µm. f , Diagram for primary culture of FRCs derived from i- Ltbr-Y / T ∆FRC-YR mice for 4 days and treatment with EtOH or 4-OHT for their analyses at 2 days after the treatment. g Comparisons of indicated mRNA expression normalized to Gapdh in primary cultured FRCs after treatment with EtOH or 4-OHT for 2 days ( n = 4). h Diagram for adipogenic culture of mesenchymal stem cells (C3H/10T1/2) infected with an adenovirus to induce overexpression of active TAZ (TAZ4SA) for their analyses at 8 days after the infection. i Immunoblot analyses of indicated proteins in mesenchymal stem cells (C3H/10T1/2) infected with an adenovirus to induce overexpression of active TAZ (TAZ4SA) or control. j Representative images of Oil Red O staining in mesenchymal stem cells (C3H/10T1/2) induced with adipogenic cocktail after infected with an adenovirus to induce overexpression of active TAZ (TAZ4SA). k Comparisons of indicated mRNA expression normalized to Gapdh in mesenchymal stem cells (C3H/10T1/2) infected with an adenovirus to induce overexpression of active TAZ (TAZ4SA) for their analyses at 2 days after the infection ( n = 4). l Schematic images proposing the importance of coordination of YAP/TAZ activity and LTβR coupling in FRCs during LN growth and maintenance. Unless otherwise denoted, horizontal bars indicate mean ± SD and P values versus non- Y / T ∆FRC-YR or non-i- Ltbr ∆FRC-YR or EtOH or Control by two‐tailed Mann‐Whitney U test.

    Techniques Used: Mouse Assay, Derivative Assay, Expressing, Cell Culture, Infection, Over Expression, Staining, Activity Assay, Two Tailed Test, MANN-WHITNEY

    YAP/TAZ support growth and structural organization of LNs by FRCs. a Diagram for generation of indicated mice and their analyses at 8 weeks after birth. b , c Representative images of YAP or TAZ in PDGFRβ + or CCL19 + FRCs in WT and Yap / Taz ∆FRC mice. FRCs around high endothelial venule (HEV) within the white dashed-line box are magnified in the lower panels with single-channel YAP or TAZ image. Scale bars, 250 µm. d Comparisons of body weight, inguinal LN weight and total number of cells within the inguinal LN in WT ( n = 11; body weight) and Yap / Taz ∆FRC mice ( n = 9; body weight). e Representative flow cytometric analysis and comparison of proportion of PDPN + CD31 − FRCs (red box) gated from CD45 − stromal cells of skin-draining LNs in WT and Yap / Taz ∆FRC mice. f Representative images and comparison of Ki-67 + FRCs (white arrows) in WT and Yap / Taz ∆FRC mice. Scale bars, 50 µm. g Comparison of indicated stromal cell counts gated from CD45 − cells of skin-draining LNs in WT and Yap / Taz ∆FRC mice. BECs ( n = 5), blood endothelial cells; LECs ( n = 6), lymphatic endothelial cells. h Representative images of distinction between B and T cells (white dashed line) beneath the LN capsule (white line) in WT and Yap / Taz ∆FRC mice. Scale bars, 200 µm. i Comparison of indicated mRNA expression in FRCs sorted from WT and Yap / Taz ∆FRC mice (quintuplicate values using n = 10–15 mice/group). j , k Representative images and comparison of DsRed + B cells and GFP + T cells within the inguinal LN at 24 h after the adoptive transfer in WT and Yap / Taz ∆FRC mice. Scale bars, 500 µm. l Changes in body weight after 1 × 10 3 pfu of A/PR/8 influenza viral infection ( n = 13). m Flow cytometric analyses and comparisons of IFN-γ+CD8 + T cells in gated CD3ε + T cells. n = 5 (CO) or 7 (IM) mice. Unless otherwise denoted, each dot indicates a value obtained from one mouse and n = 4 mice/group pooled from two independent experiments. Horizontal bars indicate mean ± SD and P values versus WT by two‐tailed Mann–Whitney U test. NS, not significant.
    Figure Legend Snippet: YAP/TAZ support growth and structural organization of LNs by FRCs. a Diagram for generation of indicated mice and their analyses at 8 weeks after birth. b , c Representative images of YAP or TAZ in PDGFRβ + or CCL19 + FRCs in WT and Yap / Taz ∆FRC mice. FRCs around high endothelial venule (HEV) within the white dashed-line box are magnified in the lower panels with single-channel YAP or TAZ image. Scale bars, 250 µm. d Comparisons of body weight, inguinal LN weight and total number of cells within the inguinal LN in WT ( n = 11; body weight) and Yap / Taz ∆FRC mice ( n = 9; body weight). e Representative flow cytometric analysis and comparison of proportion of PDPN + CD31 − FRCs (red box) gated from CD45 − stromal cells of skin-draining LNs in WT and Yap / Taz ∆FRC mice. f Representative images and comparison of Ki-67 + FRCs (white arrows) in WT and Yap / Taz ∆FRC mice. Scale bars, 50 µm. g Comparison of indicated stromal cell counts gated from CD45 − cells of skin-draining LNs in WT and Yap / Taz ∆FRC mice. BECs ( n = 5), blood endothelial cells; LECs ( n = 6), lymphatic endothelial cells. h Representative images of distinction between B and T cells (white dashed line) beneath the LN capsule (white line) in WT and Yap / Taz ∆FRC mice. Scale bars, 200 µm. i Comparison of indicated mRNA expression in FRCs sorted from WT and Yap / Taz ∆FRC mice (quintuplicate values using n = 10–15 mice/group). j , k Representative images and comparison of DsRed + B cells and GFP + T cells within the inguinal LN at 24 h after the adoptive transfer in WT and Yap / Taz ∆FRC mice. Scale bars, 500 µm. l Changes in body weight after 1 × 10 3 pfu of A/PR/8 influenza viral infection ( n = 13). m Flow cytometric analyses and comparisons of IFN-γ+CD8 + T cells in gated CD3ε + T cells. n = 5 (CO) or 7 (IM) mice. Unless otherwise denoted, each dot indicates a value obtained from one mouse and n = 4 mice/group pooled from two independent experiments. Horizontal bars indicate mean ± SD and P values versus WT by two‐tailed Mann–Whitney U test. NS, not significant.

    Techniques Used: Mouse Assay, Flow Cytometry, Expressing, Adoptive Transfer Assay, Infection, Two Tailed Test, MANN-WHITNEY

    FRC-specific depletion of Ltbr activates YAP/TAZ-induced myofibrosis. a Diagram for generation of indicated mice and their analyses at 8-weeks old. b Representative images and comparisons of indicated marker expressions on CCL19-YFP + FRCs in WT ∆FRC-YR and Ltbr ∆FRC-YR mice. Scale bars, 20 µm. c Representative images and comparisons of YAP and TAZ nuclear localization (white arrows) in inguinal LN of WT ∆FRC and Ltbr ∆FRC mice. Scale bars, 20 µm. d Comparison of indicated mRNA expression in FRCs sorted from WT ∆FRC and Ltbr ∆FRC mice. Each dot indicates a mean of quadruplicate values using n = 8–12 mice/group from three independent experiments. e Diagram for primary culture of FRCs derived from i- Lats1 / 2 ∆FRC-TR mice and treatment with EtOH (control) or 4-OHT at 4 days after the culture and their analyses at 2 days after the treatment. f Immunoblot analysis of indicated proteins in primary cultured mouse FRCs after treatment with EtOH or 4-OHT for 2 days. g Comparisons of indicated mRNA expression normalized to Gapdh in primary cultured mouse FRCs after treatment with EtOH or 4-OHT for 2 days. Each dot indicates a mean of triplicate values from three independent experiments. h , i Representative images and comparisons of indicated marker expressions in primary cultured mouse FRCs after treatment with EtOH or 4-OHT for 2 days. Scale bars, 30 μm. Each dot indicates a mean of triplicate values from three independent experiments. j Diagram for primary culture of human FRCs for 4 days and infection with an adenovirus to induce overexpression of active YAP (YAP5SA) or TAZ (TAZ4SA) for their analyses at 2 days after the infection. k , l Representative images and comparisons of indicated marker expressions in primary cultured human FRCs infected with control-, YAP5SA-, or TAZ4SA-adenovirus. Scale bars, 30 μm. Each dot indicates a mean of triplicate values from three independent experiments. Unless otherwise denoted, each dot indicates a value obtained from one mouse and n = 5 mice/group pooled from two independent experiments. Horizontal bars indicate mean ± SD and P values versus WT ∆FRC or WT ∆FRC-YR by two‐tailed Mann‐Whitney U test except for ( g ), ( i ), and ( l ) (two-tailed Student’s t -test). NS, not significant.
    Figure Legend Snippet: FRC-specific depletion of Ltbr activates YAP/TAZ-induced myofibrosis. a Diagram for generation of indicated mice and their analyses at 8-weeks old. b Representative images and comparisons of indicated marker expressions on CCL19-YFP + FRCs in WT ∆FRC-YR and Ltbr ∆FRC-YR mice. Scale bars, 20 µm. c Representative images and comparisons of YAP and TAZ nuclear localization (white arrows) in inguinal LN of WT ∆FRC and Ltbr ∆FRC mice. Scale bars, 20 µm. d Comparison of indicated mRNA expression in FRCs sorted from WT ∆FRC and Ltbr ∆FRC mice. Each dot indicates a mean of quadruplicate values using n = 8–12 mice/group from three independent experiments. e Diagram for primary culture of FRCs derived from i- Lats1 / 2 ∆FRC-TR mice and treatment with EtOH (control) or 4-OHT at 4 days after the culture and their analyses at 2 days after the treatment. f Immunoblot analysis of indicated proteins in primary cultured mouse FRCs after treatment with EtOH or 4-OHT for 2 days. g Comparisons of indicated mRNA expression normalized to Gapdh in primary cultured mouse FRCs after treatment with EtOH or 4-OHT for 2 days. Each dot indicates a mean of triplicate values from three independent experiments. h , i Representative images and comparisons of indicated marker expressions in primary cultured mouse FRCs after treatment with EtOH or 4-OHT for 2 days. Scale bars, 30 μm. Each dot indicates a mean of triplicate values from three independent experiments. j Diagram for primary culture of human FRCs for 4 days and infection with an adenovirus to induce overexpression of active YAP (YAP5SA) or TAZ (TAZ4SA) for their analyses at 2 days after the infection. k , l Representative images and comparisons of indicated marker expressions in primary cultured human FRCs infected with control-, YAP5SA-, or TAZ4SA-adenovirus. Scale bars, 30 μm. Each dot indicates a mean of triplicate values from three independent experiments. Unless otherwise denoted, each dot indicates a value obtained from one mouse and n = 5 mice/group pooled from two independent experiments. Horizontal bars indicate mean ± SD and P values versus WT ∆FRC or WT ∆FRC-YR by two‐tailed Mann‐Whitney U test except for ( g ), ( i ), and ( l ) (two-tailed Student’s t -test). NS, not significant.

    Techniques Used: Mouse Assay, Marker, Expressing, Derivative Assay, Cell Culture, Infection, Over Expression, Two Tailed Test, MANN-WHITNEY

    14) Product Images from "Lymph Node Fibroblastic Reticular Cells Construct the Stromal Reticulum via Contact with Lymphocytes"

    Article Title: Lymph Node Fibroblastic Reticular Cells Construct the Stromal Reticulum via Contact with Lymphocytes

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20040254

    Architecture of the ER-TR7 + -RN in mouse LN, and the dynamic remodeling during immune responses. (a) Whole views of LN stromal populations. Serial frozen sections were stained with several antibodies to detect CD3 (T cells), B220 (B cells), ER-TR7 antigen (reticular fibroblasts), PECAM-1 (endothelial cells), or CR1 (FDCs). The images shown are composites of multiple high-magnification images at distinct positions of an LN assembled on silica. Bar, 400 μm. (b) Higher magnification view of RN (inset in panel a) shows subreticular structures or compartments. B, B zone [follicle]; CA, capsule; HEV, high endothelial venule; MC, medullary cord; MS, medullary sinus; PCC, paracortical cord; SCS, subcapsular sinus; T, T zone. Bar, 100 μm. (c) Immunizations with OVA plus adjuvant induce dynamic remodeling of the RN within popliteal LNs. Serial frozen sections of the popliteal LNs from untreated, OVA + alum-, or OVA + CFA-immunized mice were stained with antibodies against CD3, B220, or ER-TR7. Three whole LN images are shown at the same magnification. Higher magnification view of the inset region in the medulla of CFA-immunized LN shows some nodular islands containing discrete B and T zones, enclosed by a single FRC layer (arrows). Bars: 500 μm for whole views and 100 μm for high-magnification views.
    Figure Legend Snippet: Architecture of the ER-TR7 + -RN in mouse LN, and the dynamic remodeling during immune responses. (a) Whole views of LN stromal populations. Serial frozen sections were stained with several antibodies to detect CD3 (T cells), B220 (B cells), ER-TR7 antigen (reticular fibroblasts), PECAM-1 (endothelial cells), or CR1 (FDCs). The images shown are composites of multiple high-magnification images at distinct positions of an LN assembled on silica. Bar, 400 μm. (b) Higher magnification view of RN (inset in panel a) shows subreticular structures or compartments. B, B zone [follicle]; CA, capsule; HEV, high endothelial venule; MC, medullary cord; MS, medullary sinus; PCC, paracortical cord; SCS, subcapsular sinus; T, T zone. Bar, 100 μm. (c) Immunizations with OVA plus adjuvant induce dynamic remodeling of the RN within popliteal LNs. Serial frozen sections of the popliteal LNs from untreated, OVA + alum-, or OVA + CFA-immunized mice were stained with antibodies against CD3, B220, or ER-TR7. Three whole LN images are shown at the same magnification. Higher magnification view of the inset region in the medulla of CFA-immunized LN shows some nodular islands containing discrete B and T zones, enclosed by a single FRC layer (arrows). Bars: 500 μm for whole views and 100 μm for high-magnification views.

    Techniques Used: Staining, Mass Spectrometry, Periodic Counter-current Chromatography, Mouse Assay

    15) Product Images from "Connexin37 and Connexin43 deficiencies in mice disrupt lymphatic valve development and result in lymphatic disorders including lymphedema and chylothorax"

    Article Title: Connexin37 and Connexin43 deficiencies in mice disrupt lymphatic valve development and result in lymphatic disorders including lymphedema and chylothorax

    Journal: Developmental biology

    doi: 10.1016/j.ydbio.2011.04.004

    Cx37−/−Cx43−/− embryos exhibit lymphedema and dilated superficial lymphatics
    Figure Legend Snippet: Cx37−/−Cx43−/− embryos exhibit lymphedema and dilated superficial lymphatics

    Techniques Used:

    Cx37 and Cx43 are required for lymphatic valve development in collecting vessels of the mesentery
    Figure Legend Snippet: Cx37 and Cx43 are required for lymphatic valve development in collecting vessels of the mesentery

    Techniques Used:

    Cx37+/−Cx43−/− and Cx37−/−Cx43−/− embryos display blood-filled lymphatics
    Figure Legend Snippet: Cx37+/−Cx43−/− and Cx37−/−Cx43−/− embryos display blood-filled lymphatics

    Techniques Used:

    Cx37−/−Cx43+/− mice display retrograde lymph flow and frequently die prematurely with chylothorax
    Figure Legend Snippet: Cx37−/−Cx43+/− mice display retrograde lymph flow and frequently die prematurely with chylothorax

    Techniques Used: Mouse Assay, Flow Cytometry

    Cx37, Cx43, and Cx47 become progressively enriched at mesenteric lymphatic valves and are differentially expressed in mesenteric valve leaflets
    Figure Legend Snippet: Cx37, Cx43, and Cx47 become progressively enriched at mesenteric lymphatic valves and are differentially expressed in mesenteric valve leaflets

    Techniques Used:

    Cx37, Cx43, and Cx47 are highly enriched in lymphatic valves in the adult mouse and are differentially expressed in upstream and downstream sides of thoracic duct valves
    Figure Legend Snippet: Cx37, Cx43, and Cx47 are highly enriched in lymphatic valves in the adult mouse and are differentially expressed in upstream and downstream sides of thoracic duct valves

    Techniques Used:

    Cx37−/− and Cx37−/−Cx43−/− embryos have enlarged jugular lymph sacs at E13.5
    Figure Legend Snippet: Cx37−/− and Cx37−/−Cx43−/− embryos have enlarged jugular lymph sacs at E13.5

    Techniques Used:

    Central lymphatic patterning is abnormal in Cx43−/− and Cx37−/−Cx43−/− embryos at E18.5
    Figure Legend Snippet: Central lymphatic patterning is abnormal in Cx43−/− and Cx37−/−Cx43−/− embryos at E18.5

    Techniques Used:

    Expression of Cx37, Cx43, and Cx47 in developing lymphatic vessels of the wild-type mouse embryo
    Figure Legend Snippet: Expression of Cx37, Cx43, and Cx47 in developing lymphatic vessels of the wild-type mouse embryo

    Techniques Used: Expressing

    Cx37 and Cx43 are required for thoracic duct valve development
    Figure Legend Snippet: Cx37 and Cx43 are required for thoracic duct valve development

    Techniques Used:

    16) Product Images from "Secreted NS1 of Dengue Virus Attaches to the Surface of Cells via Interactions with Heparan Sulfate and Chondroitin Sulfate E"

    Article Title: Secreted NS1 of Dengue Virus Attaches to the Surface of Cells via Interactions with Heparan Sulfate and Chondroitin Sulfate E

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0030183

    Binding of DENV NS1 to Mouse Tissues Cryo-sections of mouse (A) lung, (B) liver, and (C) intestine were incubated with serum-free supernatants from BHK DENV-2 Rep or BHK cells for 1 h at room temperature. After extensive washing, bound NS1 was detected by a mixture of NS1 mAbs (1A4, 1F11, 2G6, 1B2) followed by Cy3-conjugated goat anti-mouse IgG. Co-staining with endothelial cell marker was subsequently performed by incubating the sections with rat anti-mouse CD31 (PECAM-1) followed by Alexa Fluor 488-conjugated goat anti-rat IgG. Nuclei were stained with a DNA-specific dye TO-PRO-3. Sections incubated with DENV NS1 followed by an isotype control Ab served as a negative control. Analysis was performed by confocal microscopy. White arrow and yellow arrowhead denote the layer of endothelial cells in the lumen and the outer layer of the adventitia of pulmonary vessel, respectively.
    Figure Legend Snippet: Binding of DENV NS1 to Mouse Tissues Cryo-sections of mouse (A) lung, (B) liver, and (C) intestine were incubated with serum-free supernatants from BHK DENV-2 Rep or BHK cells for 1 h at room temperature. After extensive washing, bound NS1 was detected by a mixture of NS1 mAbs (1A4, 1F11, 2G6, 1B2) followed by Cy3-conjugated goat anti-mouse IgG. Co-staining with endothelial cell marker was subsequently performed by incubating the sections with rat anti-mouse CD31 (PECAM-1) followed by Alexa Fluor 488-conjugated goat anti-rat IgG. Nuclei were stained with a DNA-specific dye TO-PRO-3. Sections incubated with DENV NS1 followed by an isotype control Ab served as a negative control. Analysis was performed by confocal microscopy. White arrow and yellow arrowhead denote the layer of endothelial cells in the lumen and the outer layer of the adventitia of pulmonary vessel, respectively.

    Techniques Used: Binding Assay, Incubation, Staining, Marker, Negative Control, Confocal Microscopy

    Binding of DENV NS1 to Human Lung Cryo-sections of human lung tissue were incubated with 20 μg/ml of purified DENV-2 NS1 or BSA for 1 h at room temperature. Bound NS1 was detected by staining with a mixture of DENV NS1 mAbs (1A4, 1F11, 2G6, 1B2) followed by Alexa Fluor 488 conjugated with goat anti-mouse IgG. Subsequently, the sections were co-stained with rabbit anti-human CD31 (PECAM-1) followed by Cy3-conjugated donkey anti-rabbit IgG. Nuclei were stained with a DNA-specific dye (Hoechst) and the sections were analyzed by confocal microscopy. Sections incubated with purified DENV NS1 and stained with isotype mAbs served as a negative control.
    Figure Legend Snippet: Binding of DENV NS1 to Human Lung Cryo-sections of human lung tissue were incubated with 20 μg/ml of purified DENV-2 NS1 or BSA for 1 h at room temperature. Bound NS1 was detected by staining with a mixture of DENV NS1 mAbs (1A4, 1F11, 2G6, 1B2) followed by Alexa Fluor 488 conjugated with goat anti-mouse IgG. Subsequently, the sections were co-stained with rabbit anti-human CD31 (PECAM-1) followed by Cy3-conjugated donkey anti-rabbit IgG. Nuclei were stained with a DNA-specific dye (Hoechst) and the sections were analyzed by confocal microscopy. Sections incubated with purified DENV NS1 and stained with isotype mAbs served as a negative control.

    Techniques Used: Binding Assay, Incubation, Purification, Staining, Confocal Microscopy, Negative Control

    17) Product Images from "Spatial and Single-Cell Transcriptional Profiling Identifies Functionally Distinct Human Dermal Fibroblast Subpopulations"

    Article Title: Spatial and Single-Cell Transcriptional Profiling Identifies Functionally Distinct Human Dermal Fibroblast Subpopulations

    Journal: The Journal of Investigative Dermatology

    doi: 10.1016/j.jid.2018.01.016

    Human dermal fibroblast subpopulations maintain functional differences in vitro. ( a, b ) Expression of LUM and COL6A5 is enriched in CD90 + population compared with an unfractionated dermal cell suspension. Gene expression normalized to GAPDH and expressed as mean ± standard deviation for three replicates. ( c ) CD39 expression is detectable in primary CD31 – CD45 – ECad – cells but is lost after a single passage in culture ( d ). However, expression of ( d, e ) CD90 and ( e ) CD36 is retained. ( f ) lin – CD90 + CD39 + and lin – CD90 + CD36 + cells exhibit morphological differences in vitro, there is considerable intersample variation. Breast skin donor age, 19 years; abdominal skin donor age, 43 years; and thigh skin donor age, 46 years. Scale bars = 50 μm). ( g ) Quantitative PCR showing retention of LUM and loss of COL6A5 in culture. Gene expression normalized to GAPDH and expressed as mean ± standard deviation for three biological replicates. ( h–j ) Expression of genes associated with ( h ) Wnt signaling, ( i ) inflammation and immunity, and ( j ) ECM remodeling. Gene expression is normalized to 18S and is expressed as mean ± standard deviation for three biological replicates derived from female (age 42 years) abdominal skin, female (age 46 years) thigh skin, and female (age 64 years) abdominal skin. ( k–o ) Modulation of expression of cell surface markers in response to IFN-γ stimulation in culture (blue, CD39 + IFN-γ; yellow, CD36 + IFN-γ; red, unstained control). Top row: representative flow plots. Bottom row: quantitation of data from three independent experiments derived from female (age 42 years) abdominal skin, female (age 46 years) thigh skin, and female (age 64 years) abdominal skin. ∗ P
    Figure Legend Snippet: Human dermal fibroblast subpopulations maintain functional differences in vitro. ( a, b ) Expression of LUM and COL6A5 is enriched in CD90 + population compared with an unfractionated dermal cell suspension. Gene expression normalized to GAPDH and expressed as mean ± standard deviation for three replicates. ( c ) CD39 expression is detectable in primary CD31 – CD45 – ECad – cells but is lost after a single passage in culture ( d ). However, expression of ( d, e ) CD90 and ( e ) CD36 is retained. ( f ) lin – CD90 + CD39 + and lin – CD90 + CD36 + cells exhibit morphological differences in vitro, there is considerable intersample variation. Breast skin donor age, 19 years; abdominal skin donor age, 43 years; and thigh skin donor age, 46 years. Scale bars = 50 μm). ( g ) Quantitative PCR showing retention of LUM and loss of COL6A5 in culture. Gene expression normalized to GAPDH and expressed as mean ± standard deviation for three biological replicates. ( h–j ) Expression of genes associated with ( h ) Wnt signaling, ( i ) inflammation and immunity, and ( j ) ECM remodeling. Gene expression is normalized to 18S and is expressed as mean ± standard deviation for three biological replicates derived from female (age 42 years) abdominal skin, female (age 46 years) thigh skin, and female (age 64 years) abdominal skin. ( k–o ) Modulation of expression of cell surface markers in response to IFN-γ stimulation in culture (blue, CD39 + IFN-γ; yellow, CD36 + IFN-γ; red, unstained control). Top row: representative flow plots. Bottom row: quantitation of data from three independent experiments derived from female (age 42 years) abdominal skin, female (age 46 years) thigh skin, and female (age 64 years) abdominal skin. ∗ P

    Techniques Used: Functional Assay, In Vitro, Expressing, Standard Deviation, Real-time Polymerase Chain Reaction, Derivative Assay, Flow Cytometry, Quantitation Assay

    Comparison of the ability of different fibroblast subpopulations to support epidermal growth on DED. ( a ) Isolation of fibroblast subpopulations by flow cytometry. ( b–f ) Hematoxylin and eosin staining (scale bars = 100 μm) and ( g–k ) immunofluorescence staining (scale bars = 200 μm) of DED organotypic cultures ( b, g ) without fibroblasts or ( c, h ) seeded with unfractionated (lin – CD90 + ) fibroblasts, ( d, i ) CD90 + CD39 + (enriched in papillary) fibroblasts, ( e, j ) CD90 + CD39 – (depleted of papillary) fibroblasts, and ( f, k ) CD90 + CD36 + (reticular/pre-adipocyte) fibroblasts. Keratin 14 (green) marks keratinocytes, and vimentin (white) marks mesenchymal cells (fibroblasts). Experiments were repeated for a minimum of two biological replicates (donor cells derived from female [age 19 years] breast skin and female [age 37 years] breast skin; DED derived from male [age 47 years] abdominal skin, and representative images are displayed. In the case of the CD90 + CD39 + cells, two of three experiments involved additional selection for CD26 – cells to further enrich for the papillary cell population (CD90 + CD39 + CD26 – ). ( l ) Quantification of epidermal thickness, ( m ) density of fibroblasts within 300 μm of the epidermis, and ( n ) relative abundance of fibroblasts at different depths from the epidermis. adipo, adipocyte; DED, decellularized human dermis; FB, fibroblasts; FSC, forward scatter detector; K14, keratin 14; lin – , lineage negative; ns, not significant; SSC, side scatter detector.
    Figure Legend Snippet: Comparison of the ability of different fibroblast subpopulations to support epidermal growth on DED. ( a ) Isolation of fibroblast subpopulations by flow cytometry. ( b–f ) Hematoxylin and eosin staining (scale bars = 100 μm) and ( g–k ) immunofluorescence staining (scale bars = 200 μm) of DED organotypic cultures ( b, g ) without fibroblasts or ( c, h ) seeded with unfractionated (lin – CD90 + ) fibroblasts, ( d, i ) CD90 + CD39 + (enriched in papillary) fibroblasts, ( e, j ) CD90 + CD39 – (depleted of papillary) fibroblasts, and ( f, k ) CD90 + CD36 + (reticular/pre-adipocyte) fibroblasts. Keratin 14 (green) marks keratinocytes, and vimentin (white) marks mesenchymal cells (fibroblasts). Experiments were repeated for a minimum of two biological replicates (donor cells derived from female [age 19 years] breast skin and female [age 37 years] breast skin; DED derived from male [age 47 years] abdominal skin, and representative images are displayed. In the case of the CD90 + CD39 + cells, two of three experiments involved additional selection for CD26 – cells to further enrich for the papillary cell population (CD90 + CD39 + CD26 – ). ( l ) Quantification of epidermal thickness, ( m ) density of fibroblasts within 300 μm of the epidermis, and ( n ) relative abundance of fibroblasts at different depths from the epidermis. adipo, adipocyte; DED, decellularized human dermis; FB, fibroblasts; FSC, forward scatter detector; K14, keratin 14; lin – , lineage negative; ns, not significant; SSC, side scatter detector.

    Techniques Used: Isolation, Flow Cytometry, Cytometry, Staining, Immunofluorescence, Derivative Assay, Selection

    Immunofluorescence labeling of human dermis with antibodies to candidate fibroblast subpopulation markers identified by spatial transcriptomics. ( a, b ) Expression of COL6A5 is restricted to the papillary dermis (female breast skin, donor age 22 years). The basal layer of the epidermis is labeled with anti-K14 (COL6A5, green; K14, red). ( c, d ) Expression of APCDD1 is enriched in the papillary dermis (APCDD1, green; K14, red; female back skin, donor age 44 years). ( e, f ) Expression of HSPB3 is enriched in the papillary dermis (HSPB3, green; K14, red; female breast skin, donor age 22 years). ( g, h ) Expression of WIF1 is enriched in vascular structures that are more prominent in the upper dermis (WIF1, green; K14, red; female abdominal skin, donor age 27 years). ( i, j ) Expression of CD36 is highly enriched in the lower dermis (female abdominal skin, donor age 44 years). ( k, l ) CD39 is enriched in the papillary dermis (CD39, green; podoplanin, red; female abdominal skin, donor age 43 years). Scale bars = 200 μm. K14, keratin.
    Figure Legend Snippet: Immunofluorescence labeling of human dermis with antibodies to candidate fibroblast subpopulation markers identified by spatial transcriptomics. ( a, b ) Expression of COL6A5 is restricted to the papillary dermis (female breast skin, donor age 22 years). The basal layer of the epidermis is labeled with anti-K14 (COL6A5, green; K14, red). ( c, d ) Expression of APCDD1 is enriched in the papillary dermis (APCDD1, green; K14, red; female back skin, donor age 44 years). ( e, f ) Expression of HSPB3 is enriched in the papillary dermis (HSPB3, green; K14, red; female breast skin, donor age 22 years). ( g, h ) Expression of WIF1 is enriched in vascular structures that are more prominent in the upper dermis (WIF1, green; K14, red; female abdominal skin, donor age 27 years). ( i, j ) Expression of CD36 is highly enriched in the lower dermis (female abdominal skin, donor age 44 years). ( k, l ) CD39 is enriched in the papillary dermis (CD39, green; podoplanin, red; female abdominal skin, donor age 43 years). Scale bars = 200 μm. K14, keratin.

    Techniques Used: Immunofluorescence, Labeling, Expressing

    Differential expression of genes associated with Wnt, ECM, and immune signaling in mouse fibroblast subpopulations. ( a ) Gene Ontology term analysis of differentially expressed pathways in mouse fibroblast subpopulations. ( b–d ) Heat maps illustrating differential expression (Affymetrix microarray) of genes implicated in ( b ) Wnt signaling, ( c ) inflammation, and ( d ) ECM regulation. ( e ) qPCR validation of selected differentially expressed genes. ( f ) Heatmap comparing expression (Affymetrix microarray) of genes implicated in adipogenesis. ( g, h ) qPCR analysis showing up-regulation of CD36 expression in ( g ) pre-adipocyte populations and ( h ) CD39 in papillary fibroblasts. ( e, g, h ) Gene expression is normalized to GAPDH and expressed as mean ± standard deviation for three biological replicates. ∗ P
    Figure Legend Snippet: Differential expression of genes associated with Wnt, ECM, and immune signaling in mouse fibroblast subpopulations. ( a ) Gene Ontology term analysis of differentially expressed pathways in mouse fibroblast subpopulations. ( b–d ) Heat maps illustrating differential expression (Affymetrix microarray) of genes implicated in ( b ) Wnt signaling, ( c ) inflammation, and ( d ) ECM regulation. ( e ) qPCR validation of selected differentially expressed genes. ( f ) Heatmap comparing expression (Affymetrix microarray) of genes implicated in adipogenesis. ( g, h ) qPCR analysis showing up-regulation of CD36 expression in ( g ) pre-adipocyte populations and ( h ) CD39 in papillary fibroblasts. ( e, g, h ) Gene expression is normalized to GAPDH and expressed as mean ± standard deviation for three biological replicates. ∗ P

    Techniques Used: Expressing, Microarray, Real-time Polymerase Chain Reaction, Standard Deviation

    Single-cell RNA sequencing of human adult dermal fibroblasts. ( a ) Isolation of lin – cells and lin – CD90 + cells from human dermis by flow cytometry. Single live cells were isolated from a single donor (female age 64 years, abdominal skin) by gating for forward scatter, side scatter, and DAPI-staining. lin – cells were isolated by gating for CD31 – CD45 – ECad – . ( b ) PCA analysis of gene expression patterns. ( c, d ) tSNE analysis of gene expression patterns (red, lin – ; blue, lin – CD90 + ). ( d ) Automated clustering of tSNE analysis identifies five dermal fibroblast subpopulations. ( e ) Expression patterns of markers differentially expressed in each of five clusters (red, high expression; yellow, low expression. ( f ) Violin plots illustrating differential expression of marker genes in each of five dermal fibroblast subpopulations. ( g–j ) Immunostaining for candidate fibroblast markers in adult human skin: ( g ) COL6A5, female breast skin, donor age 37 years; ( h ) CD26, female breast skin, donor age 62 years; ( i ) MFAP5, female breast skin, donor age 37 years; and ( j ) RGS5, female back skin, donor age 29 years. Scale bars = 200 μm. ( k ) Isolation of lin – CD39 + CD26 – and CD39 – dermal fibroblasts by flow cytometry. ( l–p ) Expression of ( l ) CD39, ( m ) COL6A5, ( n ) WNT5A, ( o ) RSP01, and ( p ) LEF1 in lin – CD39 + CD26 – and CD39 – dermal fibroblasts. Gene expression is normalized to GAPDH and TBP and is expressed as mean ± standard deviation for three biological replicates: male (age 53 years) thigh skin, female (age 52 years) breast skin, and female (age 62 years) breast skin. ∗ P
    Figure Legend Snippet: Single-cell RNA sequencing of human adult dermal fibroblasts. ( a ) Isolation of lin – cells and lin – CD90 + cells from human dermis by flow cytometry. Single live cells were isolated from a single donor (female age 64 years, abdominal skin) by gating for forward scatter, side scatter, and DAPI-staining. lin – cells were isolated by gating for CD31 – CD45 – ECad – . ( b ) PCA analysis of gene expression patterns. ( c, d ) tSNE analysis of gene expression patterns (red, lin – ; blue, lin – CD90 + ). ( d ) Automated clustering of tSNE analysis identifies five dermal fibroblast subpopulations. ( e ) Expression patterns of markers differentially expressed in each of five clusters (red, high expression; yellow, low expression. ( f ) Violin plots illustrating differential expression of marker genes in each of five dermal fibroblast subpopulations. ( g–j ) Immunostaining for candidate fibroblast markers in adult human skin: ( g ) COL6A5, female breast skin, donor age 37 years; ( h ) CD26, female breast skin, donor age 62 years; ( i ) MFAP5, female breast skin, donor age 37 years; and ( j ) RGS5, female back skin, donor age 29 years. Scale bars = 200 μm. ( k ) Isolation of lin – CD39 + CD26 – and CD39 – dermal fibroblasts by flow cytometry. ( l–p ) Expression of ( l ) CD39, ( m ) COL6A5, ( n ) WNT5A, ( o ) RSP01, and ( p ) LEF1 in lin – CD39 + CD26 – and CD39 – dermal fibroblasts. Gene expression is normalized to GAPDH and TBP and is expressed as mean ± standard deviation for three biological replicates: male (age 53 years) thigh skin, female (age 52 years) breast skin, and female (age 62 years) breast skin. ∗ P

    Techniques Used: RNA Sequencing Assay, Isolation, Flow Cytometry, Cytometry, Staining, Expressing, Marker, Immunostaining, Standard Deviation

    18) Product Images from "Stable tumor vessel normalization with pO2 increase and endothelial PTEN activation by inositol trispyrophosphate brings novel tumor treatment"

    Article Title: Stable tumor vessel normalization with pO2 increase and endothelial PTEN activation by inositol trispyrophosphate brings novel tumor treatment

    Journal: Journal of Molecular Medicine (Berlin, Germany)

    doi: 10.1007/s00109-013-0992-6

    Effect of ITPP treatment on tumor hypoxia-induced resistance, stem cell selection, and enhancement of chemotherapeutic efficacy. a The P-glycoprotein immunostaining showing a reduced number of multidrug resistance positive tumor cells after ITPP treatment. Frozen sections of primary tumors from experiments described in Fig. 6 were histochemically labeled (day 22, ITPP treatments as described in “ Materials and methods ” ( n = 8/group; five separate experiments). Scale bars = 50 μm. b Quantification by flow cytometry showing the reduction of cells positive for precursor and stem cell-associated markers (CD133, Oct3-4, ABCG-2) after ITPP treatment. CD133 + immunostaining corroborated the reduction visible on frozen section staining of primary tumors as in a . Scale bars = 50 μm. c Lung metastasis is suppressed by chemotherapeutic drugs (Paclitaxel and Cisplatin), when treatment is preceded by ITPP injection. Tumor cells are detected by their Lucifease activity in the lungs of animals from control, ITPP, CisPt plus Paclitaxel and combined treatments ITPP + drugs as described in “ Materials and methods .” Data are reported for day 22 ( n = 10/group; 5 experiments; ***; p = 0.001). d CD31 staining of endothelial cells ( green ) and eosin/hematoxylin staining obtained in primary tumor frozen sections from experiment described in c . Efficient tissue necrosis was obtained when chemotherapeutic treatment is preceded by ITPP injection as described in “ Materials and methods .” Scale bars = 50 μm
    Figure Legend Snippet: Effect of ITPP treatment on tumor hypoxia-induced resistance, stem cell selection, and enhancement of chemotherapeutic efficacy. a The P-glycoprotein immunostaining showing a reduced number of multidrug resistance positive tumor cells after ITPP treatment. Frozen sections of primary tumors from experiments described in Fig. 6 were histochemically labeled (day 22, ITPP treatments as described in “ Materials and methods ” ( n = 8/group; five separate experiments). Scale bars = 50 μm. b Quantification by flow cytometry showing the reduction of cells positive for precursor and stem cell-associated markers (CD133, Oct3-4, ABCG-2) after ITPP treatment. CD133 + immunostaining corroborated the reduction visible on frozen section staining of primary tumors as in a . Scale bars = 50 μm. c Lung metastasis is suppressed by chemotherapeutic drugs (Paclitaxel and Cisplatin), when treatment is preceded by ITPP injection. Tumor cells are detected by their Lucifease activity in the lungs of animals from control, ITPP, CisPt plus Paclitaxel and combined treatments ITPP + drugs as described in “ Materials and methods .” Data are reported for day 22 ( n = 10/group; 5 experiments; ***; p = 0.001). d CD31 staining of endothelial cells ( green ) and eosin/hematoxylin staining obtained in primary tumor frozen sections from experiment described in c . Efficient tissue necrosis was obtained when chemotherapeutic treatment is preceded by ITPP injection as described in “ Materials and methods .” Scale bars = 50 μm

    Techniques Used: Selection, Immunostaining, Labeling, Flow Cytometry, Cytometry, Staining, Injection, Activity Assay

    19) Product Images from "Augmentation of hypoxia-inducible factor-1-alpha in reinfused blood cells enhances diabetic ischemic wound closure in mice"

    Article Title: Augmentation of hypoxia-inducible factor-1-alpha in reinfused blood cells enhances diabetic ischemic wound closure in mice

    Journal: Oncotarget

    doi: 10.18632/oncotarget.23214

    Expression of HIF-1a in re-infused BCs enhances wound-associated angiogenesis through increasing trophic macrophages (A) Representative F4/80 staining on the site of the wound. (B) Representative flow charts for analysis and sorting of F4/80+ macrophages at the site of the wound in mice. (C) RT-qPCR for F4/80 in the wound tissue. (D) RT-qPCR for VEGF-A in the sorted F4/80+ cells versus F4/80- cells from the wound tissue of the mice. * p
    Figure Legend Snippet: Expression of HIF-1a in re-infused BCs enhances wound-associated angiogenesis through increasing trophic macrophages (A) Representative F4/80 staining on the site of the wound. (B) Representative flow charts for analysis and sorting of F4/80+ macrophages at the site of the wound in mice. (C) RT-qPCR for F4/80 in the wound tissue. (D) RT-qPCR for VEGF-A in the sorted F4/80+ cells versus F4/80- cells from the wound tissue of the mice. * p

    Techniques Used: Expressing, Staining, Flow Cytometry, Mouse Assay, Quantitative RT-PCR

    20) Product Images from "Primed T Cell Responses to Chemokines Are Regulated by the Immunoglobulin-Like Molecule CD31"

    Article Title: Primed T Cell Responses to Chemokines Are Regulated by the Immunoglobulin-Like Molecule CD31

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039433

    CD31 molecules segregate differently in naïve and activated T lymphocytes. Confocal images of naive WT T cells stained with rabbit-anti-mouse CD31 (green fluorescence) and rat anti- mouse LFA-1 (red fluorescence) followed by incubation with secondary antibodies Alexa Fluor 488-conjugated donkey anti-rabbit IgG and Alexa Fluor 647-conjugated goat anti-rat IgG, respectively, are shown in panel a. Added scale bar = 6 µm. Panel b: WT Activated T cells generated via anti-CD3 and anti-CD28 treatment over 7 days were allowed to rest for 24 hours in low serum and then fixed. LFA-1 and CD31 expression was visualized as described above. Added scale bar = 6 µm. The average CD31 distribution/expression (± SD) from at least four 63× magnified fields obtained in three independent experiments of identical design is shown in panel c (*p
    Figure Legend Snippet: CD31 molecules segregate differently in naïve and activated T lymphocytes. Confocal images of naive WT T cells stained with rabbit-anti-mouse CD31 (green fluorescence) and rat anti- mouse LFA-1 (red fluorescence) followed by incubation with secondary antibodies Alexa Fluor 488-conjugated donkey anti-rabbit IgG and Alexa Fluor 647-conjugated goat anti-rat IgG, respectively, are shown in panel a. Added scale bar = 6 µm. Panel b: WT Activated T cells generated via anti-CD3 and anti-CD28 treatment over 7 days were allowed to rest for 24 hours in low serum and then fixed. LFA-1 and CD31 expression was visualized as described above. Added scale bar = 6 µm. The average CD31 distribution/expression (± SD) from at least four 63× magnified fields obtained in three independent experiments of identical design is shown in panel c (*p

    Techniques Used: Staining, Fluorescence, Incubation, Generated, Expressing

    Polarization of CD31 molecules in activated T cells following exposure to chemokines. Activated WT T cells were allowed to migrate through transwells in response to the chemokine CXCL-10 (panel a) or incubated in medium alone (panel b) and then fixed for analysis. Cells were not permeabilized to allow surface staining only. Confocal images of T cells stained with rabbit-anti-mouse CD31 followed by incubation with Alexa Fluor 488-conjugated donkey anti-rabbit IgG are shown in panels a and b. A higher magnification of a migrating lymphocyte further depicting CD31 molecule polarization is shown in panel c. Confocal z stacks series were acquired using a step size of 0.5 µm. Added scale bar = 6 µm. The average CD31 distribution/expression (± SD) from at least four 63× magnified fields obtained in three independent experiments of identical design is shown in panel b (*p
    Figure Legend Snippet: Polarization of CD31 molecules in activated T cells following exposure to chemokines. Activated WT T cells were allowed to migrate through transwells in response to the chemokine CXCL-10 (panel a) or incubated in medium alone (panel b) and then fixed for analysis. Cells were not permeabilized to allow surface staining only. Confocal images of T cells stained with rabbit-anti-mouse CD31 followed by incubation with Alexa Fluor 488-conjugated donkey anti-rabbit IgG are shown in panels a and b. A higher magnification of a migrating lymphocyte further depicting CD31 molecule polarization is shown in panel c. Confocal z stacks series were acquired using a step size of 0.5 µm. Added scale bar = 6 µm. The average CD31 distribution/expression (± SD) from at least four 63× magnified fields obtained in three independent experiments of identical design is shown in panel b (*p

    Techniques Used: Incubation, Staining, Expressing

    CD31 inhibits chemokine-induced Akt phosphorylation in activated T cells. Naïve and activated T cells were exposed to CCL19/21 and CXCL10, respectively, for 2 minutes. Phosphorylation of Akt at serine 473 was assessed by antibody staining and flow cytometry. In panels a and c. representative histograms of the experimental conditions indicated beside each profile are shown. Panel b and d indicate cumulative data of the mean fluorescence intensity (MFI) indicative of Akt phosphorylation obtained in the various conditions indicated in at least 4 independent experiments of identical design. *p
    Figure Legend Snippet: CD31 inhibits chemokine-induced Akt phosphorylation in activated T cells. Naïve and activated T cells were exposed to CCL19/21 and CXCL10, respectively, for 2 minutes. Phosphorylation of Akt at serine 473 was assessed by antibody staining and flow cytometry. In panels a and c. representative histograms of the experimental conditions indicated beside each profile are shown. Panel b and d indicate cumulative data of the mean fluorescence intensity (MFI) indicative of Akt phosphorylation obtained in the various conditions indicated in at least 4 independent experiments of identical design. *p

    Techniques Used: Staining, Flow Cytometry, Cytometry, Fluorescence

    CD31-deficient T cell chemokinesis is partially resistant to PI3K inhibition. Naïve (panel a) and activated (panel b) WT and CD31−/−T cell migration in response to either CCL19/21 (naïve) or CXCL10 (activated) through a transwell was assessed as described in Methods . Some T cells were pre-incubated with the PI3K inhibitor Wortmannin (10 µM) for 30 minutes at RT. Percentage migration was calculated by dividing the number of cells harvested from the bottom chamber following 6 hours incubation at 37°C by the original number of cells plated onto the transwell. The average percentage migration from at least three independent experiments is shown. Error bars indicate SD (*p
    Figure Legend Snippet: CD31-deficient T cell chemokinesis is partially resistant to PI3K inhibition. Naïve (panel a) and activated (panel b) WT and CD31−/−T cell migration in response to either CCL19/21 (naïve) or CXCL10 (activated) through a transwell was assessed as described in Methods . Some T cells were pre-incubated with the PI3K inhibitor Wortmannin (10 µM) for 30 minutes at RT. Percentage migration was calculated by dividing the number of cells harvested from the bottom chamber following 6 hours incubation at 37°C by the original number of cells plated onto the transwell. The average percentage migration from at least three independent experiments is shown. Error bars indicate SD (*p

    Techniques Used: Inhibition, Migration, Incubation

    CD31-deficient activated T cells display enhanced responses to chemokines in vitro and in vivo . Panels a-b: Naïve and activated WT and CD31−/− T cell migration through a transwell in response to the chemokines CCL19/21 and CXCL10, respectively, was assessed over 6 hours. Percentage migration was calculated by dividing the number of cells in the bottom chamber by the original number of cells plated onto the transwell. The average percentage migration from four independent experiments is shown. Error bars indicate SD (*p
    Figure Legend Snippet: CD31-deficient activated T cells display enhanced responses to chemokines in vitro and in vivo . Panels a-b: Naïve and activated WT and CD31−/− T cell migration through a transwell in response to the chemokines CCL19/21 and CXCL10, respectively, was assessed over 6 hours. Percentage migration was calculated by dividing the number of cells in the bottom chamber by the original number of cells plated onto the transwell. The average percentage migration from four independent experiments is shown. Error bars indicate SD (*p

    Techniques Used: In Vitro, In Vivo, Migration

    Antibody-mediated CD31 ‘immobilization’ enhances activated T cell chemokinesis. Naïve (a) and activated (b) WT T cells were incubated overnight in RPMI 0.5% FCS. Some T cells were pre-incubated with an anti-CD31 mAb at saturating concentrations (5 µg/ml) for 30 minutes at RT. Migration in response to CXCL10 or medium through a transwell was assessed over 6 hours. Percentage migration was calculated by dividing the number of cells harvested from the bottom chamber following 6 hours incubation at 37°C by the original number of cells plated onto the transwell. The average percentage migration from three independent experiments is shown. Error bars indicate SD (*p
    Figure Legend Snippet: Antibody-mediated CD31 ‘immobilization’ enhances activated T cell chemokinesis. Naïve (a) and activated (b) WT T cells were incubated overnight in RPMI 0.5% FCS. Some T cells were pre-incubated with an anti-CD31 mAb at saturating concentrations (5 µg/ml) for 30 minutes at RT. Migration in response to CXCL10 or medium through a transwell was assessed over 6 hours. Percentage migration was calculated by dividing the number of cells harvested from the bottom chamber following 6 hours incubation at 37°C by the original number of cells plated onto the transwell. The average percentage migration from three independent experiments is shown. Error bars indicate SD (*p

    Techniques Used: Incubation, Migration

    21) Product Images from "P-Selectin Glycoprotein Ligand-1 Deficiency Is Protective Against Obesity-Related Insulin Resistance"

    Article Title: P-Selectin Glycoprotein Ligand-1 Deficiency Is Protective Against Obesity-Related Insulin Resistance

    Journal: Diabetes

    doi: 10.2337/db09-1894

    A : Liver weight ( left ) and hepatic triglyceride ( right ) in WT mice (□) and PSGL-1 −/− (KO) mice (■) fed HFD for 10 weeks ( n = 5 [WT-HF]; n = 8 [KO-HF]). Data are means ± SE. * P
    Figure Legend Snippet: A : Liver weight ( left ) and hepatic triglyceride ( right ) in WT mice (□) and PSGL-1 −/− (KO) mice (■) fed HFD for 10 weeks ( n = 5 [WT-HF]; n = 8 [KO-HF]). Data are means ± SE. * P

    Techniques Used: Mouse Assay

    A : Periodical acid Schiff staining of epididymal fat sections from WT mice ( left-hand panel ) and PSGL-1 −/− (KO) mice ( right-hand panel ) fed HFD for 10 weeks. The scale bars represent 100 μm. B : Distribution of adipocyte size in epididymal fat tissues from WT mice (□) and PSGL-1 −/− mice (■). Data are the mean from analysis of six high-power fields from each of five mice. C : Immunohistochemical detection of Mac-3 in epididymal fat tissue from WT mice ( upper panel s) and PSGL-1 −/− mice ( lower panel s) fed HFD. Macrophage infiltration into epididymal fat tissue decreased in PSGL-1 −/− mice. Scale bars, 50 μm. D : Gene expression of F4/80, CD11c, IL-10, MCP-1, IL-6, iNOS, leptin, and LPL in epididymal fat tissues from WT mice (□) and PSGL-1 −/− mice (■) fed HFD analyzed by quantitative real-time RT-PCR ( n = 7 [WT-HF]; n = 7 [KO-HF]). Data are means ± SE. * P
    Figure Legend Snippet: A : Periodical acid Schiff staining of epididymal fat sections from WT mice ( left-hand panel ) and PSGL-1 −/− (KO) mice ( right-hand panel ) fed HFD for 10 weeks. The scale bars represent 100 μm. B : Distribution of adipocyte size in epididymal fat tissues from WT mice (□) and PSGL-1 −/− mice (■). Data are the mean from analysis of six high-power fields from each of five mice. C : Immunohistochemical detection of Mac-3 in epididymal fat tissue from WT mice ( upper panel s) and PSGL-1 −/− mice ( lower panel s) fed HFD. Macrophage infiltration into epididymal fat tissue decreased in PSGL-1 −/− mice. Scale bars, 50 μm. D : Gene expression of F4/80, CD11c, IL-10, MCP-1, IL-6, iNOS, leptin, and LPL in epididymal fat tissues from WT mice (□) and PSGL-1 −/− mice (■) fed HFD analyzed by quantitative real-time RT-PCR ( n = 7 [WT-HF]; n = 7 [KO-HF]). Data are means ± SE. * P

    Techniques Used: Staining, Mouse Assay, Immunohistochemistry, Expressing, Quantitative RT-PCR

    A : Immunohistochemical localization of PSGL-1, macrophages, and endothelial cells in adipose tissue. Epididymal fat pads from 8-week-old db / db mice and WT mice were stained with anti–MAC-3 ( left-hand panels ) and anti–PSGL-1 antibodies ( right-hand panels ). Macrophages and PSGL-1 expressed around the small vessels in the interstitium of adipose tissue in db / db mice are shown. The scale bars represent 50 μm. B : Double immunofluorescence staining of adipose tissue from db / db mice with the antibodies against PSGL-1 ( green ) and leukocyte (CD45, red ). PSGL-1 and CD45 were stained in the interstitium of adipose tissue and are colocalized in the merged picture. C : Double immunofluorescence staining of adipose tissue from db / db mice with the antibodies against PSGL-1 ( green ) and endothelial cell (CD31, red ). PSGL-1 and CD31 were stained along small vessels of adipose tissue and are colocalized in the merged picture. D–F : The expression of PSGL-1 on cells in WT mice and db / db mice was analyzed using flow cytometry. D : The expression of PSGL-1 in PBMCs. E : The expression of PSGL-1 in F4/80 + macrophages in the SVF from adipose tissue. F : The expression of PSGL-1 in CD31 + endothelial cells in the SVF from adipose tissue. (A high-quality digital representation of this figure is available in the online issue.)
    Figure Legend Snippet: A : Immunohistochemical localization of PSGL-1, macrophages, and endothelial cells in adipose tissue. Epididymal fat pads from 8-week-old db / db mice and WT mice were stained with anti–MAC-3 ( left-hand panels ) and anti–PSGL-1 antibodies ( right-hand panels ). Macrophages and PSGL-1 expressed around the small vessels in the interstitium of adipose tissue in db / db mice are shown. The scale bars represent 50 μm. B : Double immunofluorescence staining of adipose tissue from db / db mice with the antibodies against PSGL-1 ( green ) and leukocyte (CD45, red ). PSGL-1 and CD45 were stained in the interstitium of adipose tissue and are colocalized in the merged picture. C : Double immunofluorescence staining of adipose tissue from db / db mice with the antibodies against PSGL-1 ( green ) and endothelial cell (CD31, red ). PSGL-1 and CD31 were stained along small vessels of adipose tissue and are colocalized in the merged picture. D–F : The expression of PSGL-1 on cells in WT mice and db / db mice was analyzed using flow cytometry. D : The expression of PSGL-1 in PBMCs. E : The expression of PSGL-1 in F4/80 + macrophages in the SVF from adipose tissue. F : The expression of PSGL-1 in CD31 + endothelial cells in the SVF from adipose tissue. (A high-quality digital representation of this figure is available in the online issue.)

    Techniques Used: Immunohistochemistry, Mouse Assay, Staining, Double Immunofluorescence Staining, Expressing, Flow Cytometry, Cytometry

    A : Metabolic characteristics of WT mice and PSGL-1 −/− (KO) mice fed HFD from 7 to 17 weeks old. Body composition and food intake in WT mice (□) and PSGL-1 −/− mice (■) fed HFD ( n = 7 [WT-HF]; n = 8 [KO-HF]) is shown. B : Metabolic parameters of WT mice (□) and PSGL-1 −/− mice (■) fed HFD ( n = 7 [WT-HF]; n = 8 [KO-HF]). C : Blood glucose level ( upper panel ) and plasma insulin levels ( lower panel ) during the glucose tolerance test (1.2 g/kg body mass) ( n = 9 [WT-HF], ○; n = 8 [KO-HF], ●). D : Blood glucose level during the insulin tolerance test (0.7 units/kg body mass) ( n = 9 [WT-HF], ○; n = 8 [KO-HF], ●). Data are means ± SE. * P
    Figure Legend Snippet: A : Metabolic characteristics of WT mice and PSGL-1 −/− (KO) mice fed HFD from 7 to 17 weeks old. Body composition and food intake in WT mice (□) and PSGL-1 −/− mice (■) fed HFD ( n = 7 [WT-HF]; n = 8 [KO-HF]) is shown. B : Metabolic parameters of WT mice (□) and PSGL-1 −/− mice (■) fed HFD ( n = 7 [WT-HF]; n = 8 [KO-HF]). C : Blood glucose level ( upper panel ) and plasma insulin levels ( lower panel ) during the glucose tolerance test (1.2 g/kg body mass) ( n = 9 [WT-HF], ○; n = 8 [KO-HF], ●). D : Blood glucose level during the insulin tolerance test (0.7 units/kg body mass) ( n = 9 [WT-HF], ○; n = 8 [KO-HF], ●). Data are means ± SE. * P

    Techniques Used: Mouse Assay

    22) Product Images from "CD206+ M2-like macrophages regulate systemic glucose metabolism by inhibiting proliferation of adipocyte progenitors"

    Article Title: CD206+ M2-like macrophages regulate systemic glucose metabolism by inhibiting proliferation of adipocyte progenitors

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00231-1

    Depletion of CD206 + M2-like macrophages promote browning of iWAT. a Relative mRNA expression of UCP1, PGC-1α. CPT-1β and CD137 levels in iWAT after CD206 + cells depletion under cold exposure ( n = 3–5 mice per group). b Immunostaining of paraffin section of eWAT stained with anti-UCP1 antibody in DT-treated CD206DTR mice after cold exposure compared with WT control. The images were taken with a Leica TCS-SP5 (40×). c Representative flow cytometry analysis of PDGFRα/Sca-1 double positive cells from iWAT of DT-treated CD206DTR and WT mice at RT and cold stimulated (25 °C and 6 °C) (left panel). The percentage of Lin(−)APs positive fraction in the DT-treated CD206DTR mice are shown in the right panel ( n = 3–5). For this, first, negative selection of PE-Cy7 anti CD31 (endothelial), FITC anti-mouse lineage cocktail were selected followed by positive selection of PE anti-PDGFRα and APC-Cy7 anti Sca-1. Full gating strategy is given in Supplementary Fig. 13 b.The data are shown as the means ± SEM. * P
    Figure Legend Snippet: Depletion of CD206 + M2-like macrophages promote browning of iWAT. a Relative mRNA expression of UCP1, PGC-1α. CPT-1β and CD137 levels in iWAT after CD206 + cells depletion under cold exposure ( n = 3–5 mice per group). b Immunostaining of paraffin section of eWAT stained with anti-UCP1 antibody in DT-treated CD206DTR mice after cold exposure compared with WT control. The images were taken with a Leica TCS-SP5 (40×). c Representative flow cytometry analysis of PDGFRα/Sca-1 double positive cells from iWAT of DT-treated CD206DTR and WT mice at RT and cold stimulated (25 °C and 6 °C) (left panel). The percentage of Lin(−)APs positive fraction in the DT-treated CD206DTR mice are shown in the right panel ( n = 3–5). For this, first, negative selection of PE-Cy7 anti CD31 (endothelial), FITC anti-mouse lineage cocktail were selected followed by positive selection of PE anti-PDGFRα and APC-Cy7 anti Sca-1. Full gating strategy is given in Supplementary Fig. 13 b.The data are shown as the means ± SEM. * P

    Techniques Used: Expressing, Pyrolysis Gas Chromatography, Cycling Probe Technology, Mouse Assay, Immunostaining, Paraffin Section, Staining, Flow Cytometry, Cytometry, Selection

    23) Product Images from "RUNX transcription factors potentially control E-selectin expression in the bone marrow vascular niche in mice"

    Article Title: RUNX transcription factors potentially control E-selectin expression in the bone marrow vascular niche in mice

    Journal: Blood Advances

    doi: 10.1182/bloodadvances.2017009324

    Inhibition of RUNX1 attenuates the E-selectin expression. (A) Relative expression of E-selectin , P-selectin , VCAM1 , TIE2 , SDF1 ( CXCL12 ), ICAM1 , and JAG1 was determined in HUVECs treated with control DMSO or Chb-M′ at 0.5 μM or 1.0 μM. Twenty-four hours after treatment, total RNA was prepared and processed for real-time qPCR analysis. Values are normalized to those of DMSO-treated cells (n = 3). (B) Correlation between the expression levels of RUNX1 and E-selectin in HUVEC cell lines established from 9 different umbilical cords (n = 9). Values represent array signal intensities. P values were determined using Spearman’s correlation. (C) Surface expressions of E-selectin were determined in HUVEC treated either by control DMSO or Chb-M′ at 1 μM. Forty-eight hours after treatment, cells were harvested and stained for flow cytometric analysis. (D) Relative expression of E-selectin , P-selectin , VCAM1 , TIE2 , SDF1 ( CXCL12 ), ICAM1 , and JAG1 was determined in HUVECs transduced with control (sh_ Luc .) or RUNX1 shRNA (sh_ RUNX1 ) in the presence of 3 μM doxycycline. Twenty-four hours after treatment, total RNA was prepared and processed for real-time RT-PCR analysis. Values are normalized to that of control cells (n = 3). (E) Surface expression of E-selectin was determined in HUVECs transduced with sh_ Luc . or sh_ RUNX1 in the presence of 3 μM doxycycline. Forty-eight hours after treatment, cells were harvested and stained for flow cytometric analysis. (F) Schematic illustration showing the proximal regulatory region (−600 bp to +200 bp of the transcription start site) of E-selectin . (G) ChIP analysis in HUVECs using anti-RUNX1 antibody, an isotope-matched control IgG, and anti-histone H3 antibody. ChIP products were subjected to PCR-based amplification with the indicated primer sets (supplemental Table 1), using RPL30 as a negative control. (H) Schematic representation of the treatment and analysis schedule in C57BL/6 mice. Mice were treated with andrographolide (25 mg/kg body weight, 3 times/week, IP), A 205804 (10 mg/kg body weight, 3 times/week, orally [p.o.]), or Chb-M′ (320 μg/kg body weight, 3 times/week, IV) for 2 weeks. After treatment, mice were sacrificed, and endosteal cells were dislodged from the femurs for further analysis. (I) Representative flow cytometry analysis identifying vascular niche cells in the endosteal cells obtained in panel H. Expression of E-selectin was determined in the indicated endothelial cells (Lin − CD45 − CD31 + ). (J) Representative flow cytometry analysis of E-selectin expression in endothelial cells in mice treated with control DMSO or andrographolide. (K) Representative flow cytometry analysis of E-selectin expression in endothelial cells in mice treated with control DMSO or A 205804. (L) Representative flow cytometry analysis of E-selectin expression in endothelial cells in mice treated with control DMSO or Chb-M′. Data are presented as mean ± SEM. * P
    Figure Legend Snippet: Inhibition of RUNX1 attenuates the E-selectin expression. (A) Relative expression of E-selectin , P-selectin , VCAM1 , TIE2 , SDF1 ( CXCL12 ), ICAM1 , and JAG1 was determined in HUVECs treated with control DMSO or Chb-M′ at 0.5 μM or 1.0 μM. Twenty-four hours after treatment, total RNA was prepared and processed for real-time qPCR analysis. Values are normalized to those of DMSO-treated cells (n = 3). (B) Correlation between the expression levels of RUNX1 and E-selectin in HUVEC cell lines established from 9 different umbilical cords (n = 9). Values represent array signal intensities. P values were determined using Spearman’s correlation. (C) Surface expressions of E-selectin were determined in HUVEC treated either by control DMSO or Chb-M′ at 1 μM. Forty-eight hours after treatment, cells were harvested and stained for flow cytometric analysis. (D) Relative expression of E-selectin , P-selectin , VCAM1 , TIE2 , SDF1 ( CXCL12 ), ICAM1 , and JAG1 was determined in HUVECs transduced with control (sh_ Luc .) or RUNX1 shRNA (sh_ RUNX1 ) in the presence of 3 μM doxycycline. Twenty-four hours after treatment, total RNA was prepared and processed for real-time RT-PCR analysis. Values are normalized to that of control cells (n = 3). (E) Surface expression of E-selectin was determined in HUVECs transduced with sh_ Luc . or sh_ RUNX1 in the presence of 3 μM doxycycline. Forty-eight hours after treatment, cells were harvested and stained for flow cytometric analysis. (F) Schematic illustration showing the proximal regulatory region (−600 bp to +200 bp of the transcription start site) of E-selectin . (G) ChIP analysis in HUVECs using anti-RUNX1 antibody, an isotope-matched control IgG, and anti-histone H3 antibody. ChIP products were subjected to PCR-based amplification with the indicated primer sets (supplemental Table 1), using RPL30 as a negative control. (H) Schematic representation of the treatment and analysis schedule in C57BL/6 mice. Mice were treated with andrographolide (25 mg/kg body weight, 3 times/week, IP), A 205804 (10 mg/kg body weight, 3 times/week, orally [p.o.]), or Chb-M′ (320 μg/kg body weight, 3 times/week, IV) for 2 weeks. After treatment, mice were sacrificed, and endosteal cells were dislodged from the femurs for further analysis. (I) Representative flow cytometry analysis identifying vascular niche cells in the endosteal cells obtained in panel H. Expression of E-selectin was determined in the indicated endothelial cells (Lin − CD45 − CD31 + ). (J) Representative flow cytometry analysis of E-selectin expression in endothelial cells in mice treated with control DMSO or andrographolide. (K) Representative flow cytometry analysis of E-selectin expression in endothelial cells in mice treated with control DMSO or A 205804. (L) Representative flow cytometry analysis of E-selectin expression in endothelial cells in mice treated with control DMSO or Chb-M′. Data are presented as mean ± SEM. * P

    Techniques Used: Inhibition, Expressing, Real-time Polymerase Chain Reaction, Staining, Flow Cytometry, Transduction, shRNA, Quantitative RT-PCR, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Negative Control, Mouse Assay, Cytometry

    24) Product Images from "Three-dimensional cartography of hematopoietic clusters in the vasculature of whole mouse embryos"

    Article Title: Three-dimensional cartography of hematopoietic clusters in the vasculature of whole mouse embryos

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.051094

    Functional analyses of enriched mouse hematopoietic cluster cells. ( A ) FACS profile of the caudal half region, VA and UA cells at E10 (31-34) sp. SSEA1 – cells were sorted into four populations: R1 (endothelial cells, 0.37±0.12%), R2 (hematopoietic clusters, 0.081±0.009%), R3 (0.10±0.03%) and R4 (75.5±6.0%). ( B ) Methylcellulose colony-forming assay of sorted cells from 31-34 sp embryos. CFU-GEMM, CFU-G/M/GM and BFU-E were counted after 7-8 days of culture. Results are mean ± s.d. of three independent experiments. ( C ) HSC activity in hematopoietic clusters. * Secondary repopulation was also observed. ** AGM equivalent. VA, vitelline artery; UA, umbilical artery; PB, peripheral blood. ( D ) Representative hematopoietic multi-lineage FACS profile of long-term reconstituted mice. Peripheral blood was analyzed 4 months post-injection.
    Figure Legend Snippet: Functional analyses of enriched mouse hematopoietic cluster cells. ( A ) FACS profile of the caudal half region, VA and UA cells at E10 (31-34) sp. SSEA1 – cells were sorted into four populations: R1 (endothelial cells, 0.37±0.12%), R2 (hematopoietic clusters, 0.081±0.009%), R3 (0.10±0.03%) and R4 (75.5±6.0%). ( B ) Methylcellulose colony-forming assay of sorted cells from 31-34 sp embryos. CFU-GEMM, CFU-G/M/GM and BFU-E were counted after 7-8 days of culture. Results are mean ± s.d. of three independent experiments. ( C ) HSC activity in hematopoietic clusters. * Secondary repopulation was also observed. ** AGM equivalent. VA, vitelline artery; UA, umbilical artery; PB, peripheral blood. ( D ) Representative hematopoietic multi-lineage FACS profile of long-term reconstituted mice. Peripheral blood was analyzed 4 months post-injection.

    Techniques Used: Functional Assay, FACS, Activity Assay, Mouse Assay, Injection

    Flow cytometric enrichment of mouse hematopoietic cluster cells. ( A , B ) FACS analysis of the caudal half region and of VA and UA cells at E10.5 (34-36 sp). (B) Red and blue dots in the right panel indicate macrophages and PGCs, respectively. No/few of these cells are present in the cluster gate. ( C ) Whole-mount immunostaining for c-Kit, CD31 and SSEA1 expression at E10.5 (36 sp). c-Kit lo CD31 + SSEA1 + PGCs are localized ventrolaterally under aorta. Movie 4 in the supplementary material shows a 3D image. Scale bar: 100 μm. Graph shows relative intensity of c-Kit staining on cluster cells and SSEA1 + PGCs. ( D ) Quantification of PGCs and clusters in the caudal half region, and in the VA and UA using immunofluorescence and FACS analysis at E10.5 (34-36 sp). Average number per embryo is indicated. Numerous c-Kit + CD31 + cells are present in small vessels connected to VA (not shown) and are included in this quantification. ( E ) CD45 and Flk1 expression within the cluster gate. Most CD45 hi cells (red box, middle panel) within the cluster gate are Flk1 negative (red dots, right panel). ( F ) Hematopoietic cluster stained with anti CD45, CD31 and Flk1 antibodies. ( G ) Whole-mount immunostaining of Runx1 –/– embryo for c-Kit and CD31 expression. Scale bar: 100 μm. No c-Kit + cells were observed in Runx1 –/– aorta. Movie 5 in the supplementary material shows a 3D image. Graph shows the number of c-Kit + cells part dorsal aorta (DA) from Runx1 +/+ (E10.5, 35-37 sp) and Runx1 –/– (E10.5, 35 and 36 sp); n =3. ( H ) FACS analysis of Runx1 +/+ and Runx1 –/– embryos. Cluster gate cells were absent in Runx1 –/– embryos. Six Runx1 –/– embryos (E10, 28-34 sp) analyzed gave consistent data. VA, vitelline artery.
    Figure Legend Snippet: Flow cytometric enrichment of mouse hematopoietic cluster cells. ( A , B ) FACS analysis of the caudal half region and of VA and UA cells at E10.5 (34-36 sp). (B) Red and blue dots in the right panel indicate macrophages and PGCs, respectively. No/few of these cells are present in the cluster gate. ( C ) Whole-mount immunostaining for c-Kit, CD31 and SSEA1 expression at E10.5 (36 sp). c-Kit lo CD31 + SSEA1 + PGCs are localized ventrolaterally under aorta. Movie 4 in the supplementary material shows a 3D image. Scale bar: 100 μm. Graph shows relative intensity of c-Kit staining on cluster cells and SSEA1 + PGCs. ( D ) Quantification of PGCs and clusters in the caudal half region, and in the VA and UA using immunofluorescence and FACS analysis at E10.5 (34-36 sp). Average number per embryo is indicated. Numerous c-Kit + CD31 + cells are present in small vessels connected to VA (not shown) and are included in this quantification. ( E ) CD45 and Flk1 expression within the cluster gate. Most CD45 hi cells (red box, middle panel) within the cluster gate are Flk1 negative (red dots, right panel). ( F ) Hematopoietic cluster stained with anti CD45, CD31 and Flk1 antibodies. ( G ) Whole-mount immunostaining of Runx1 –/– embryo for c-Kit and CD31 expression. Scale bar: 100 μm. No c-Kit + cells were observed in Runx1 –/– aorta. Movie 5 in the supplementary material shows a 3D image. Graph shows the number of c-Kit + cells part dorsal aorta (DA) from Runx1 +/+ (E10.5, 35-37 sp) and Runx1 –/– (E10.5, 35 and 36 sp); n =3. ( H ) FACS analysis of Runx1 +/+ and Runx1 –/– embryos. Cluster gate cells were absent in Runx1 –/– embryos. Six Runx1 –/– embryos (E10, 28-34 sp) analyzed gave consistent data. VA, vitelline artery.

    Techniques Used: Flow Cytometry, FACS, Immunostaining, Expressing, Staining, Immunofluorescence

    25) Product Images from "anlotinib alters tumor immune microenvironment by downregulating PD-L1 expression on vascular endothelial cells"

    Article Title: anlotinib alters tumor immune microenvironment by downregulating PD-L1 expression on vascular endothelial cells

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-020-2511-3

    A schematic representation of effects of PD-L1 expression on VECs in tumor microenvironment. a Pattern diagram of tumor immune microenvironment. b PD-L1 highly expressed on VECs in tumor tissues inhibits activation of CD8 + T cells and promotes immune escape of tumors. c anlotinib inhibits the expression of PD-L1 on VECs through the AKT pathway. d anlotinib improved the ratio of CD8/FoxP3 and broke the immune barrier by inhibiting the VEC-PD-L1.
    Figure Legend Snippet: A schematic representation of effects of PD-L1 expression on VECs in tumor microenvironment. a Pattern diagram of tumor immune microenvironment. b PD-L1 highly expressed on VECs in tumor tissues inhibits activation of CD8 + T cells and promotes immune escape of tumors. c anlotinib inhibits the expression of PD-L1 on VECs through the AKT pathway. d anlotinib improved the ratio of CD8/FoxP3 and broke the immune barrier by inhibiting the VEC-PD-L1.

    Techniques Used: Expressing, Activation Assay

    anlotinib improves the immune microenvironment via increasing the ratio of CD8/FoxP3. a Representative flow images of CD4 + CD25 + FoxP3 + T cells taken from B16 tumors treated as above indicated. b , c Statistics of the percentage of CD4 + T cells, CD8 + T cells, FoxP3 + T cells and ratio of CD8/FoxP3 in the B16 or MC38 tumors. d Representative images of CD31 immunostaining (red), 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining (blue) and markers of immune cells (CD4, CD8, and FoxP3) (green) of B16 tumors ( n = 5 per group, except for Bev group n = 4) treated as above indicated. Scale bars,100 mm. e Quantification of CD4 + T cells, CD8 + T cells, FoxP3 + T cells in B16 tumors. f Statistics of perivascular and without perivascular CD8 + T cells in the B16 tumors. g Statistics of ratio of CD8/FoxP3 in the B16 or MC38 tumors. All data are exhibited as mean ± SD. Statistical differences were assessed using the unpaired Student’s test. * P
    Figure Legend Snippet: anlotinib improves the immune microenvironment via increasing the ratio of CD8/FoxP3. a Representative flow images of CD4 + CD25 + FoxP3 + T cells taken from B16 tumors treated as above indicated. b , c Statistics of the percentage of CD4 + T cells, CD8 + T cells, FoxP3 + T cells and ratio of CD8/FoxP3 in the B16 or MC38 tumors. d Representative images of CD31 immunostaining (red), 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining (blue) and markers of immune cells (CD4, CD8, and FoxP3) (green) of B16 tumors ( n = 5 per group, except for Bev group n = 4) treated as above indicated. Scale bars,100 mm. e Quantification of CD4 + T cells, CD8 + T cells, FoxP3 + T cells in B16 tumors. f Statistics of perivascular and without perivascular CD8 + T cells in the B16 tumors. g Statistics of ratio of CD8/FoxP3 in the B16 or MC38 tumors. All data are exhibited as mean ± SD. Statistical differences were assessed using the unpaired Student’s test. * P

    Techniques Used: Immunostaining, Staining

    Endothelial PD-L1 expression affects the efficacy of anlotinib. a C57BL/6 mice were injected with 1 × 10 6 B16 cells and tumors grew. On day 12, mice were divided into four groups ( n = 8 per group), and treated with bEnd.3-vector, bEnd.3-CD274, PBS or anlotinib as shown. b Representative images of B16 tumor tissues treated as above indicated. c Left: tumor growth curve of the various treatment groups; right: Statistics of the weight of B16 tumors treated as indicated. d Representative flow images of CD4 + CD25 + FoxP3 + T cells taken from B16 tumors treated as indicated. e Statistics of the percentage of CD8 + T cells, CD8 + IFN-γ + T cells, FoxP3 + T cells and ratio of CD8/FoxP3 in the tumors. Data are mean ± SD. * P
    Figure Legend Snippet: Endothelial PD-L1 expression affects the efficacy of anlotinib. a C57BL/6 mice were injected with 1 × 10 6 B16 cells and tumors grew. On day 12, mice were divided into four groups ( n = 8 per group), and treated with bEnd.3-vector, bEnd.3-CD274, PBS or anlotinib as shown. b Representative images of B16 tumor tissues treated as above indicated. c Left: tumor growth curve of the various treatment groups; right: Statistics of the weight of B16 tumors treated as indicated. d Representative flow images of CD4 + CD25 + FoxP3 + T cells taken from B16 tumors treated as indicated. e Statistics of the percentage of CD8 + T cells, CD8 + IFN-γ + T cells, FoxP3 + T cells and ratio of CD8/FoxP3 in the tumors. Data are mean ± SD. * P

    Techniques Used: Expressing, Mouse Assay, Injection, Plasmid Preparation

    PD-L1 high expression on VECs is closely related to the infiltration of immune cells. a , c Representative images of CD8 + T cells and FoxP3 + T cells in sections taken from subjects with either PD-L1 + or PD-L1 − vessels in lung, kidney, and colon cancer. Original magnification, ×200. The number of intratumoral CD8 + T cells and FoxP3 + T cells from patients’ sections (kidney or colon cancer) associated with the percentage of PD-L1 + vessels. The determination as high and low were grouped by the median values. Lung adenocarcinoma ( n = 41) b , colon cancer ( n = 50) e and kidney cancer ( n = 32) d ; scale bars: 100 µm. Data are mean ± SD. * P
    Figure Legend Snippet: PD-L1 high expression on VECs is closely related to the infiltration of immune cells. a , c Representative images of CD8 + T cells and FoxP3 + T cells in sections taken from subjects with either PD-L1 + or PD-L1 − vessels in lung, kidney, and colon cancer. Original magnification, ×200. The number of intratumoral CD8 + T cells and FoxP3 + T cells from patients’ sections (kidney or colon cancer) associated with the percentage of PD-L1 + vessels. The determination as high and low were grouped by the median values. Lung adenocarcinoma ( n = 41) b , colon cancer ( n = 50) e and kidney cancer ( n = 32) d ; scale bars: 100 µm. Data are mean ± SD. * P

    Techniques Used: Expressing

    PD-L1 expression on endothelial cells affects the proliferation and activation of immune cells. a PBMC was extracted from normal blood and treated with 2.5 µg/ml anti-CD3 antibody and IL-2 (100 U/ml) for 72 h, and co-cultured with tumor-derived endothelial cells (Td-EC) for 48 h. b HUVECs were treated with the supernatant of tumor cells (CM) for 48 h. c PBMC and Td-EC were treated with control, anlotinib (0.1 μ m ) or anti-PD-L1 antibody (4 μg/ml). d Detection of the CD8 + IFN-γ + , CD8 + Ki67 + , CD8 + GranzymeB + , and FoxP3 + T cells by flow cytometry. e Quantification of the above positive cells in the immune cells. f Quantification of cytokine levels in co-culture supernatants (2 days) by liquid microarray. Data are mean ± SD. * P
    Figure Legend Snippet: PD-L1 expression on endothelial cells affects the proliferation and activation of immune cells. a PBMC was extracted from normal blood and treated with 2.5 µg/ml anti-CD3 antibody and IL-2 (100 U/ml) for 72 h, and co-cultured with tumor-derived endothelial cells (Td-EC) for 48 h. b HUVECs were treated with the supernatant of tumor cells (CM) for 48 h. c PBMC and Td-EC were treated with control, anlotinib (0.1 μ m ) or anti-PD-L1 antibody (4 μg/ml). d Detection of the CD8 + IFN-γ + , CD8 + Ki67 + , CD8 + GranzymeB + , and FoxP3 + T cells by flow cytometry. e Quantification of the above positive cells in the immune cells. f Quantification of cytokine levels in co-culture supernatants (2 days) by liquid microarray. Data are mean ± SD. * P

    Techniques Used: Expressing, Activation Assay, Cell Culture, Derivative Assay, Flow Cytometry, Co-Culture Assay, Microarray

    26) Product Images from "Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity"

    Article Title: Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity

    Journal: Nature

    doi: 10.1038/nature24302

    IgA + plasmocytes regulate tumour killing by CD8 + T cells a , Dih10 and dihXY HCC cells were transfected with an inducible ovalbumin (Ova) expression vector, and Ova expression and presentation were confirmed by flow cytometry, using an antibody that recognized the SIINFEKL peptide on the MHCI molecule H-2Kb. b – h , Ova-expressing dih cells or controls (dih–RFP) were starved overnight (5% cell death), after which their medium was changed and B cells from WT, Iga −/− , Pdl1/2 −/− , or SW-HEL mice were added in the presence of TGFβ (5 ng ml −1 ) and CTGF (3 ng ml −1 ), for an additional 24 h. Thereafter, the medium was replaced and CFSE-labelled OT-I T cells were added to the cultures that either contained or did not contain the B cells described above. After 4–6 days, the co-cultured cells were analysed by flow cytometry, while the secretory IgA was analysed by ELISA ( n = 2–4 wells per group per day). a – j , Experiments were repeated with two different Ova-expressing HCC and one prostate cancer cell lines. Shown are the representative flow cytometry histograms or plots depicting ( b ) OT-I CD8 + T-cell proliferation, ( c ) PD-L1 and SIINFEKL/H-2Kb expression on cancer cells, ( d ) PD-L1 expression on B cells, ( e ) cancer cell death. f , Relative dih-Ova–RFP killing by OT-I CD8 + T cells in the presence or absence of the indicated B cells. g , Total secretory IgA and anti-OVA-IgA antibody amounts in culture supernatants. h , Percentages of OT-I CD8 + cells in each culture, as indicated. i , j , TRC2-Ova–RFP cells or its control cell line (TRC2–RFP) were co-cultured with OT-I cells and splenic B cells (WT and Il10 −/− ), as described for a – h. i , Proliferation of OT-I cells was analysed using CFSE ( n = 3, 7, 5, 4). j , The amounts of secretory IgA were analysed using ELISA as indicated ( n = 3 per group). k – o , Liver cells from indicated 3-month-old mice were stained and analysed by flow cytometry. Experiments were repeated at least two or three times. Each dot represents one mouse. Shown are the percentage of CD8 + T cells among CD45 + cells ( n = 6, 5, 3, 7, 4) ( k ), absolute CD8 + T-cell number per gram of liver ( n = 3, 3, 7, 4) ( l ), the percentage of CD8 + CD44 + Ki-67 + T cells with representative scatter plots ( n = 3 or 4 per group) ( m , n ), and the representative scatter plots of perforin and GrzB among CD8 + CD44 + Ki-67 + T cells ( o ). p , q , Liver cell suspensions from the indicated mice were stained as shown and analysed by flow cytometry to determine the absolute CD8 + T-cell number in both STAM-BL6 and STAM-FVB mice ( n = 8, 4, 5, 3, 5, 5, 6, 7) ( p ), and the percentage of T EM cells using CD8, CD44, and CD62L ( n = 4, 4, 6, 9) ( q ). r , Liver cells from indicated 3-, 6-, and 11-month-old mice kept on HFD ( n = 4–10) were stained and analysed by flow cytometry. Shown are the percentage of CD8 + IFNγ + CD107a + T cells. Detailed n values are shown in . s – y , Liver cell suspensions from the indicated mice were stained as shown and analysed by flow cytometry to determine the percentage of Th17 cells using CD4 and IL-17a ( n = 4, 7, 9, 10, 4, 9, 11) ( s ), the percentage of regulatory T cells using CD4 and Foxp3 ( n = 4, 3, 8, 11, 7, 13) ( t ), the percentage of Tfh-like cells using CXCR5, PD1 and CD4 (3, 5, 6, 5) ( u ), the percentage of B220 + CD19 + B cells ( n = 4, 7, 7, 4, 5, 3, 9, 3, 11) ( v ), absolute B220 + CD19 + B-cell number per gram of liver ( n = 5, 7, 12, 27, 15, 7) ( w ), the percentage of IgG + cells ( n = 4, 3, 14, 11) ( x ), and CD138 + plasma cells ( n = 4, 7, 4, 7, 7, 8, 5, 8, 6, 6, 13, 27, 17) ( y ). Two-sided t -test (means ± s.e.m.; f – m ) and Mann–Whitney test (median; p – y ) were used to determine significance. * P
    Figure Legend Snippet: IgA + plasmocytes regulate tumour killing by CD8 + T cells a , Dih10 and dihXY HCC cells were transfected with an inducible ovalbumin (Ova) expression vector, and Ova expression and presentation were confirmed by flow cytometry, using an antibody that recognized the SIINFEKL peptide on the MHCI molecule H-2Kb. b – h , Ova-expressing dih cells or controls (dih–RFP) were starved overnight (5% cell death), after which their medium was changed and B cells from WT, Iga −/− , Pdl1/2 −/− , or SW-HEL mice were added in the presence of TGFβ (5 ng ml −1 ) and CTGF (3 ng ml −1 ), for an additional 24 h. Thereafter, the medium was replaced and CFSE-labelled OT-I T cells were added to the cultures that either contained or did not contain the B cells described above. After 4–6 days, the co-cultured cells were analysed by flow cytometry, while the secretory IgA was analysed by ELISA ( n = 2–4 wells per group per day). a – j , Experiments were repeated with two different Ova-expressing HCC and one prostate cancer cell lines. Shown are the representative flow cytometry histograms or plots depicting ( b ) OT-I CD8 + T-cell proliferation, ( c ) PD-L1 and SIINFEKL/H-2Kb expression on cancer cells, ( d ) PD-L1 expression on B cells, ( e ) cancer cell death. f , Relative dih-Ova–RFP killing by OT-I CD8 + T cells in the presence or absence of the indicated B cells. g , Total secretory IgA and anti-OVA-IgA antibody amounts in culture supernatants. h , Percentages of OT-I CD8 + cells in each culture, as indicated. i , j , TRC2-Ova–RFP cells or its control cell line (TRC2–RFP) were co-cultured with OT-I cells and splenic B cells (WT and Il10 −/− ), as described for a – h. i , Proliferation of OT-I cells was analysed using CFSE ( n = 3, 7, 5, 4). j , The amounts of secretory IgA were analysed using ELISA as indicated ( n = 3 per group). k – o , Liver cells from indicated 3-month-old mice were stained and analysed by flow cytometry. Experiments were repeated at least two or three times. Each dot represents one mouse. Shown are the percentage of CD8 + T cells among CD45 + cells ( n = 6, 5, 3, 7, 4) ( k ), absolute CD8 + T-cell number per gram of liver ( n = 3, 3, 7, 4) ( l ), the percentage of CD8 + CD44 + Ki-67 + T cells with representative scatter plots ( n = 3 or 4 per group) ( m , n ), and the representative scatter plots of perforin and GrzB among CD8 + CD44 + Ki-67 + T cells ( o ). p , q , Liver cell suspensions from the indicated mice were stained as shown and analysed by flow cytometry to determine the absolute CD8 + T-cell number in both STAM-BL6 and STAM-FVB mice ( n = 8, 4, 5, 3, 5, 5, 6, 7) ( p ), and the percentage of T EM cells using CD8, CD44, and CD62L ( n = 4, 4, 6, 9) ( q ). r , Liver cells from indicated 3-, 6-, and 11-month-old mice kept on HFD ( n = 4–10) were stained and analysed by flow cytometry. Shown are the percentage of CD8 + IFNγ + CD107a + T cells. Detailed n values are shown in . s – y , Liver cell suspensions from the indicated mice were stained as shown and analysed by flow cytometry to determine the percentage of Th17 cells using CD4 and IL-17a ( n = 4, 7, 9, 10, 4, 9, 11) ( s ), the percentage of regulatory T cells using CD4 and Foxp3 ( n = 4, 3, 8, 11, 7, 13) ( t ), the percentage of Tfh-like cells using CXCR5, PD1 and CD4 (3, 5, 6, 5) ( u ), the percentage of B220 + CD19 + B cells ( n = 4, 7, 7, 4, 5, 3, 9, 3, 11) ( v ), absolute B220 + CD19 + B-cell number per gram of liver ( n = 5, 7, 12, 27, 15, 7) ( w ), the percentage of IgG + cells ( n = 4, 3, 14, 11) ( x ), and CD138 + plasma cells ( n = 4, 7, 4, 7, 7, 8, 5, 8, 6, 6, 13, 27, 17) ( y ). Two-sided t -test (means ± s.e.m.; f – m ) and Mann–Whitney test (median; p – y ) were used to determine significance. * P

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Flow Cytometry, Cytometry, Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, MANN-WHITNEY

    27) Product Images from "Depletion of white adipocyte progenitors induces beige adipocyte differentiation and suppresses obesity development"

    Article Title: Depletion of white adipocyte progenitors induces beige adipocyte differentiation and suppresses obesity development

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2014.148

    WAP depletion suppresses WAT growth in DIO mice. Mice (15/group) maintained on high-fat diet underwent a 4-week treatment: twelve 200 μ l s.c. injections (3/week) of 1 mM D-WAT (▪) or PBS (control; ). ( a ) Changes in total, fat, and lean body mass during and after treatment. ( b ) Exposed i.p. WAT and intradermal s.c. WAT (bracketed in H E-stained sections) hypotrophic in treated mice at week 8. WAT pad weights are quantified in the graph on the right. ( c ) Phase contrast micrographs of adherent cells recovered (week 8) from matched amount of i.p. WAT, interscapular BAT, and lung tissue showing reduced plating efficiency for WAT, but not for BAT or lung, of treated mice. Arrows indicate SVF cells with ASC morphology, arrowheads indicate monocytes, and asterisks indicate endothelial colonies. ( d ) Identification of mesenchymal stroma as CD31−CD45− cells with large nuclei (arrows) versus leukocytes as CD45+ cells (arrowheads) and endothelial cells as CD31+ (*) by immunofluorescence on adherent cells shown in ( c ) with anti-CD31 (red) and anti-CD45 (green) antibodies. ( e ) Quantification of SVF immunophenotyping ( d ) showing reduced frequency of ASCs among viable WAT cells. ( f ) The i.p. WAT cells shown in ( c ) post confluence were induced to differentiate into white adipocytes (7 days) and stained with Oil red O. Note that white adipocytes (accumulating large lipid droplets) are rare upon treatment. Error bars: S.E.M. * P
    Figure Legend Snippet: WAP depletion suppresses WAT growth in DIO mice. Mice (15/group) maintained on high-fat diet underwent a 4-week treatment: twelve 200 μ l s.c. injections (3/week) of 1 mM D-WAT (▪) or PBS (control; ). ( a ) Changes in total, fat, and lean body mass during and after treatment. ( b ) Exposed i.p. WAT and intradermal s.c. WAT (bracketed in H E-stained sections) hypotrophic in treated mice at week 8. WAT pad weights are quantified in the graph on the right. ( c ) Phase contrast micrographs of adherent cells recovered (week 8) from matched amount of i.p. WAT, interscapular BAT, and lung tissue showing reduced plating efficiency for WAT, but not for BAT or lung, of treated mice. Arrows indicate SVF cells with ASC morphology, arrowheads indicate monocytes, and asterisks indicate endothelial colonies. ( d ) Identification of mesenchymal stroma as CD31−CD45− cells with large nuclei (arrows) versus leukocytes as CD45+ cells (arrowheads) and endothelial cells as CD31+ (*) by immunofluorescence on adherent cells shown in ( c ) with anti-CD31 (red) and anti-CD45 (green) antibodies. ( e ) Quantification of SVF immunophenotyping ( d ) showing reduced frequency of ASCs among viable WAT cells. ( f ) The i.p. WAT cells shown in ( c ) post confluence were induced to differentiate into white adipocytes (7 days) and stained with Oil red O. Note that white adipocytes (accumulating large lipid droplets) are rare upon treatment. Error bars: S.E.M. * P

    Techniques Used: Mouse Assay, Staining, Immunofluorescence

    D-WAT peptide induces ASC apoptosis. ( a ) Adherent ASCs from i.p. WAT were incubated with Cy3-labeled peptide WAT7 (red), washed, and subjected to anti-CD44 confocal immunofluorescence visualizing cell surface (green). Intracellular peptide (arrow) is indicated in Z -stack projections of median series. ( b ) Flow cytometric quantification of Apo-Trace uptake demonstrates i.p. SVF apoptosis induced by 0.05 mM D-WAT treatment; Control: untreated cells. ( c ) Phase contrast micrographs of s.c. SVF after 0.05 mM D-WAT treatment shows dead (Trypan blue positive) ASCs among viable monocytes (arrowheads) and endothelial cells (*). Plot on the right shows D-WAT dose dependence measured based on Trypan blue+ cell frequency. ( d ) Micrographs of indicated cells demonstrate apoptosis induced by D-WAT but not by the uncoupled d WAT7 and d KLAKLAK 2 domains or by Adipotide (CKGGRAKDC- d KLAKLAK 2 ). Note D-WAT sensitivity of s.c. and i.p. ASCs but not of BAT-derived stroma. ( e and f ) Mice were s.c. injected with D-WAT or PBS (Control), and 48 h later tissues were analyzed. ( e ) Upon subsequent i.v. Apo-Trace injection, in vivo apoptosis was detected through flow cytometric analysis of identical cell numbers for control and treated tissues demonstrates D-WAT-induced apoptosis in s.c. and i.p. WAT, but not in BAT. ( f ) D-WAT dose-dependent apoptosis frequency observed for cells from s.c. and i.p. WAT, but not for control organs. ( g ) Flow cytometric analysis of matched numbers of s.c. and i.p. SVF from mice injected with D-WAT in ( e ) with PE-conjugated CD31 antibody and APC-Cy7-conjugated CD45 antibody demonstrates a higher frequency of leukocytes (CD45+CD31−) in i.p. WAT. ( h ) Immunofluorescence on sections of indicated tissues from D-WAT-treated mice with antibody against cleaved Caspase 3 (green) demonstrates apoptosis (arrows) in s.c. WAT perivascular cells. ( i ) The s.c. WAT section immunofluorescence with anti-decorin antibody (green) demonstrates loss of perivascular DCN-expressing cells (arrows) specifically in treated mice. Endothelium is stained with anti-CD31 antibody (red). Blue: nuclei. Scale bar: 50 μ m
    Figure Legend Snippet: D-WAT peptide induces ASC apoptosis. ( a ) Adherent ASCs from i.p. WAT were incubated with Cy3-labeled peptide WAT7 (red), washed, and subjected to anti-CD44 confocal immunofluorescence visualizing cell surface (green). Intracellular peptide (arrow) is indicated in Z -stack projections of median series. ( b ) Flow cytometric quantification of Apo-Trace uptake demonstrates i.p. SVF apoptosis induced by 0.05 mM D-WAT treatment; Control: untreated cells. ( c ) Phase contrast micrographs of s.c. SVF after 0.05 mM D-WAT treatment shows dead (Trypan blue positive) ASCs among viable monocytes (arrowheads) and endothelial cells (*). Plot on the right shows D-WAT dose dependence measured based on Trypan blue+ cell frequency. ( d ) Micrographs of indicated cells demonstrate apoptosis induced by D-WAT but not by the uncoupled d WAT7 and d KLAKLAK 2 domains or by Adipotide (CKGGRAKDC- d KLAKLAK 2 ). Note D-WAT sensitivity of s.c. and i.p. ASCs but not of BAT-derived stroma. ( e and f ) Mice were s.c. injected with D-WAT or PBS (Control), and 48 h later tissues were analyzed. ( e ) Upon subsequent i.v. Apo-Trace injection, in vivo apoptosis was detected through flow cytometric analysis of identical cell numbers for control and treated tissues demonstrates D-WAT-induced apoptosis in s.c. and i.p. WAT, but not in BAT. ( f ) D-WAT dose-dependent apoptosis frequency observed for cells from s.c. and i.p. WAT, but not for control organs. ( g ) Flow cytometric analysis of matched numbers of s.c. and i.p. SVF from mice injected with D-WAT in ( e ) with PE-conjugated CD31 antibody and APC-Cy7-conjugated CD45 antibody demonstrates a higher frequency of leukocytes (CD45+CD31−) in i.p. WAT. ( h ) Immunofluorescence on sections of indicated tissues from D-WAT-treated mice with antibody against cleaved Caspase 3 (green) demonstrates apoptosis (arrows) in s.c. WAT perivascular cells. ( i ) The s.c. WAT section immunofluorescence with anti-decorin antibody (green) demonstrates loss of perivascular DCN-expressing cells (arrows) specifically in treated mice. Endothelium is stained with anti-CD31 antibody (red). Blue: nuclei. Scale bar: 50 μ m

    Techniques Used: Incubation, Labeling, Immunofluorescence, Flow Cytometry, Derivative Assay, Mouse Assay, Injection, In Vivo, Expressing, Staining

    28) Product Images from "CD8+ T-cell density imaging with 64Cu-labeled cys-diabody informs immunotherapy protocols"

    Article Title: CD8+ T-cell density imaging with 64Cu-labeled cys-diabody informs immunotherapy protocols

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    doi: 10.1158/1078-0432.CCR-18-0261

    PET/CT image, image-derived accumulation of 64 Cu-169cDb and results of validation by flow cytometry, each in wild-type mice. ( A ) Projected small animal PET/CT images of wild-type FVB (left), BALB/c (middle), and C57BL/6 (right) mouse acquired at 24 hours after intravenous administration of 64 Cu-TETA-169cDb (3.7 ± 0.30 mg) through the tail vein. (LN: Lymph Node (white arrows), S: Spleen, K: Kidney, L: Liver, and I: intestine (yellow arrow)). Image intensity is scaled as the maximum 30% ID/cc. ( B ) Biodistribution of 64 ). (C) The number of T-cells (CD45 + CD3 + cells), (D) the fraction of T-cells (CD45 + CD3 + cells given as a percentage of live cells) and (E) the number of CD8 + T cells. Data, mean ± SD (unpaired t test with Welch’s correction, *** P
    Figure Legend Snippet: PET/CT image, image-derived accumulation of 64 Cu-169cDb and results of validation by flow cytometry, each in wild-type mice. ( A ) Projected small animal PET/CT images of wild-type FVB (left), BALB/c (middle), and C57BL/6 (right) mouse acquired at 24 hours after intravenous administration of 64 Cu-TETA-169cDb (3.7 ± 0.30 mg) through the tail vein. (LN: Lymph Node (white arrows), S: Spleen, K: Kidney, L: Liver, and I: intestine (yellow arrow)). Image intensity is scaled as the maximum 30% ID/cc. ( B ) Biodistribution of 64 ). (C) The number of T-cells (CD45 + CD3 + cells), (D) the fraction of T-cells (CD45 + CD3 + cells given as a percentage of live cells) and (E) the number of CD8 + T cells. Data, mean ± SD (unpaired t test with Welch’s correction, *** P

    Techniques Used: Positron Emission Tomography, Derivative Assay, Flow Cytometry, Cytometry, Mouse Assay

    29) Product Images from "The endothelial antigen ESAM marks primitive hematopoietic progenitors throughout life in mice"

    Article Title: The endothelial antigen ESAM marks primitive hematopoietic progenitors throughout life in mice

    Journal: Blood

    doi: 10.1182/blood-2008-07-167106

    Specific expression of ESAM on HSC-enriched fraction of E14.5 fetal liver . Flow cytometric analysis was performed for Rag1/GFP − cells of E14.5 fetal liver using anti-c-kit, anti-Sca1, and anti-ESAM Abs. First, Rag1/GFP − cells were sorted from E14.5 fetal liver of Rag1/GFP knockin heterozygous fetuses with high purity. The sorted cells were incubated with a purified rat anti–mouse ESAM Ab (1G8) followed by goat anti–rat IgG-FITC. The cells were then stained with anti-c-kit-APC, anti-Sca1-PE, and 7-AAD. To minimize the nonspecific binding of anti-c-kit and Sca1 mAbs to the cells wearing goat anti–rat IgG-FITC, the cells were incubated with a rat anti–mouse FcRII/III Ab before the anti-c-kit and Sca1 staining. (A) The Rag1/GFP − cells were analyzed with respect to expression of ESAM, c-kit, and Sca1 (Left, middle). Expression of c-kit in the Sca1 Hi ESAM Hi cells (middle, inset) is presented (right). (B) The conventional c-kit Hi Sca1 + fraction (left, inset) could be divided into 2 fractions, ESAM −/Lo and ESAM Hi (middle). The cells were stained with an isotype control IgG (dashed line) or with the anti-ESAM Ab (solid line). The ESAM Hi cells (yellow) were found as c-kit Hi Sca1 Hi , whereas the ESAM −/Lo cells (pink) were c-kit Hi Sca1 Lo (right). (C) Six-color flow cytometric analysis using an anti-ESAM Ab followed by goat anti–rat IgG-FITC, a PE-anti-CD48 Ab, biotin-anti-lineage marker Abs (TER119, Gr1, CD3, CD45R/B220) followed by SA-PETR, a PE-Cy7-anti-Sca1 Ab, an APC-anti-c-kit, and 7-AAD was performed for E14.5 fetal liver cells of WT C57B6 embryos. The profile of Lin − cells regarding c-kit and Sca1 expression is shown in the left. The Lin − c-kit Hi Sca1 Lo and Lin − c-kit Hi Sca1 Hi fractions gated in the left panel were analyzed with respect to expression of ESAM and CD48 (middle and right). The percentage of cells in each gate is indicated in each panel.
    Figure Legend Snippet: Specific expression of ESAM on HSC-enriched fraction of E14.5 fetal liver . Flow cytometric analysis was performed for Rag1/GFP − cells of E14.5 fetal liver using anti-c-kit, anti-Sca1, and anti-ESAM Abs. First, Rag1/GFP − cells were sorted from E14.5 fetal liver of Rag1/GFP knockin heterozygous fetuses with high purity. The sorted cells were incubated with a purified rat anti–mouse ESAM Ab (1G8) followed by goat anti–rat IgG-FITC. The cells were then stained with anti-c-kit-APC, anti-Sca1-PE, and 7-AAD. To minimize the nonspecific binding of anti-c-kit and Sca1 mAbs to the cells wearing goat anti–rat IgG-FITC, the cells were incubated with a rat anti–mouse FcRII/III Ab before the anti-c-kit and Sca1 staining. (A) The Rag1/GFP − cells were analyzed with respect to expression of ESAM, c-kit, and Sca1 (Left, middle). Expression of c-kit in the Sca1 Hi ESAM Hi cells (middle, inset) is presented (right). (B) The conventional c-kit Hi Sca1 + fraction (left, inset) could be divided into 2 fractions, ESAM −/Lo and ESAM Hi (middle). The cells were stained with an isotype control IgG (dashed line) or with the anti-ESAM Ab (solid line). The ESAM Hi cells (yellow) were found as c-kit Hi Sca1 Hi , whereas the ESAM −/Lo cells (pink) were c-kit Hi Sca1 Lo (right). (C) Six-color flow cytometric analysis using an anti-ESAM Ab followed by goat anti–rat IgG-FITC, a PE-anti-CD48 Ab, biotin-anti-lineage marker Abs (TER119, Gr1, CD3, CD45R/B220) followed by SA-PETR, a PE-Cy7-anti-Sca1 Ab, an APC-anti-c-kit, and 7-AAD was performed for E14.5 fetal liver cells of WT C57B6 embryos. The profile of Lin − cells regarding c-kit and Sca1 expression is shown in the left. The Lin − c-kit Hi Sca1 Lo and Lin − c-kit Hi Sca1 Hi fractions gated in the left panel were analyzed with respect to expression of ESAM and CD48 (middle and right). The percentage of cells in each gate is indicated in each panel.

    Techniques Used: Expressing, Flow Cytometry, Knock-In, Incubation, Purification, Staining, Binding Assay, Marker

    30) Product Images from "A Novel Fully-Human Potency-Matched Dual Cytokine-Antibody Fusion Protein Targets Carbonic Anhydrase IX in Renal Cell Carcinomas"

    Article Title: A Novel Fully-Human Potency-Matched Dual Cytokine-Antibody Fusion Protein Targets Carbonic Anhydrase IX in Renal Cell Carcinomas

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2019.01228

    Antigen expression and tumor targeting properties of IL2-XE114-TNF mut . Microscopic fluorescence analysis of CAIX expression on SKRC52 tumor sections ( A,B upper left) detected with IL2-XE114-TNF mut or IL2-KSF-TNF mut (negative control). Microscopic fluorescence analysis of organs from SKRC52 tumor bearing mice, 24 h after intravenous administration of IL2-XE114-TNF mut (A) or IL2-KSF-TNF mut (B) . Cryosections were stained with anti-IL2 (green, AlexaFluor 488) and anti-CD31 (red, AlexaFluor 594). 20x magnification, scale bars = 100 μm.
    Figure Legend Snippet: Antigen expression and tumor targeting properties of IL2-XE114-TNF mut . Microscopic fluorescence analysis of CAIX expression on SKRC52 tumor sections ( A,B upper left) detected with IL2-XE114-TNF mut or IL2-KSF-TNF mut (negative control). Microscopic fluorescence analysis of organs from SKRC52 tumor bearing mice, 24 h after intravenous administration of IL2-XE114-TNF mut (A) or IL2-KSF-TNF mut (B) . Cryosections were stained with anti-IL2 (green, AlexaFluor 488) and anti-CD31 (red, AlexaFluor 594). 20x magnification, scale bars = 100 μm.

    Techniques Used: Expressing, Fluorescence, Negative Control, Mouse Assay, Staining

    Biochemical characterization of IL2-XE114-TNF mut . (A) Schematic representation of the domain assembly of IL2-XE114-TNF mut in non-covalent homotrimeric format. (B) Size exclusion chromatography profile. (C) BIAcore analysis on CAIX-coated sensor chip. (D) ESI-MS profile. (E) IL2 bioactivity assay on CTLL2 cells. (F) TNF bioactivity assay on L-M fibroblasts. (G) SDS-PAGE analysis; MW, molecular weight; NR, non-reducing conditions; R, reducing conditions. (H) Flow cytometric evaluation of CAIX expression by SKRC52 cells, detected with IL2-XE114-TNF mut . (I) Differential Scanning Fluorimetry on IL2-XE114-TNF mut .
    Figure Legend Snippet: Biochemical characterization of IL2-XE114-TNF mut . (A) Schematic representation of the domain assembly of IL2-XE114-TNF mut in non-covalent homotrimeric format. (B) Size exclusion chromatography profile. (C) BIAcore analysis on CAIX-coated sensor chip. (D) ESI-MS profile. (E) IL2 bioactivity assay on CTLL2 cells. (F) TNF bioactivity assay on L-M fibroblasts. (G) SDS-PAGE analysis; MW, molecular weight; NR, non-reducing conditions; R, reducing conditions. (H) Flow cytometric evaluation of CAIX expression by SKRC52 cells, detected with IL2-XE114-TNF mut . (I) Differential Scanning Fluorimetry on IL2-XE114-TNF mut .

    Techniques Used: Size-exclusion Chromatography, Chromatin Immunoprecipitation, Mass Spectrometry, SDS Page, Molecular Weight, Flow Cytometry, Expressing

    31) Product Images from "Mammary molecular portraits reveal lineage-specific features and progenitor cell vulnerabilities"

    Article Title: Mammary molecular portraits reveal lineage-specific features and progenitor cell vulnerabilities

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201804042

    DAC and JQ1 prevent adult progenitor cell expansion and mammopoiesis in vivo. (A) Workflow schematic for in vivo drug testing. (B and C) Flow cytometry analysis of luminal (CD24 + CD49f lo ) and basal (CD24 − CD49f hi ) mammary subsets. Primary mammary cells were purified from the two inguinal glands of mice treated for 1 wk with vehicle, JQ1 (gray background), or DAC (clear background), + progesterone. Left: Bar charts show absolute number of ER − PR − basal, ER − PR − luminal progenitor, and ER + PR + luminal cells, which were further purified using the CD49b and SCA-1 cell-surface markers. Right: Bar charts show absolute number of CFC. Error bars for all bar charts represent SEM. Statistical significance was calculated using one-way (absolute CFC) or two-way (absolute ER − PR − basal, ER − PR − luminal progenitor, and ER + PR + luminal) ANOVA followed by Dunnett’s multiple comparisons test. All multiple comparisons testing was performed with a 0.05 significance level and 95% confidence interval; statistically significant differences are indicated by asterisks, which denote size of significance levels. In B, biological replicates: n ≥ 7 left stacked bar charts, n ≥ 4 right CFC bar chart; in C, biological replicates: n ≥ 4. (D) Representative whole mounts from mice treated with vehicle or indicated epigenetic drug. Bars, 1 mm. (E) IF staining of mammary ductal structures: DAPI (blue), basal lineage marker KRT14 (red), and DNMT1 (green). Luminal/basal border is depicted by a white dotted line. Bars, 20 µm. (F) Bar charts show relative frequency of primary mammary dead cells purified from the two inguinal glands of mice treated for 1 wk with JQ1 (gray background) or DAC, + progesterone. Dead cells were determined via propidium iodide staining; biological replicates: n ≥7 (JQ1) or n ≥4 (DAC). Statistical significance was tested for using one-way ANOVA followed by Dunnett’s multiple comparisons test; no comparisons were found to be statistically significant. (G) Bar charts show relative frequency of early- and late-apoptotic mammary cells after treatment with 0.5 mg/kg DAC, three weekly doses for 4 wk, determined via annexin V and propidium iodide staining. Biological replicates, n = 5. Statistical significance was tested using two-way ANOVA followed by Sidak’s multiple comparison test; no comparisons were found to be statistically significant. *, P ≤ 0.05; **, P ≤ 0.01.
    Figure Legend Snippet: DAC and JQ1 prevent adult progenitor cell expansion and mammopoiesis in vivo. (A) Workflow schematic for in vivo drug testing. (B and C) Flow cytometry analysis of luminal (CD24 + CD49f lo ) and basal (CD24 − CD49f hi ) mammary subsets. Primary mammary cells were purified from the two inguinal glands of mice treated for 1 wk with vehicle, JQ1 (gray background), or DAC (clear background), + progesterone. Left: Bar charts show absolute number of ER − PR − basal, ER − PR − luminal progenitor, and ER + PR + luminal cells, which were further purified using the CD49b and SCA-1 cell-surface markers. Right: Bar charts show absolute number of CFC. Error bars for all bar charts represent SEM. Statistical significance was calculated using one-way (absolute CFC) or two-way (absolute ER − PR − basal, ER − PR − luminal progenitor, and ER + PR + luminal) ANOVA followed by Dunnett’s multiple comparisons test. All multiple comparisons testing was performed with a 0.05 significance level and 95% confidence interval; statistically significant differences are indicated by asterisks, which denote size of significance levels. In B, biological replicates: n ≥ 7 left stacked bar charts, n ≥ 4 right CFC bar chart; in C, biological replicates: n ≥ 4. (D) Representative whole mounts from mice treated with vehicle or indicated epigenetic drug. Bars, 1 mm. (E) IF staining of mammary ductal structures: DAPI (blue), basal lineage marker KRT14 (red), and DNMT1 (green). Luminal/basal border is depicted by a white dotted line. Bars, 20 µm. (F) Bar charts show relative frequency of primary mammary dead cells purified from the two inguinal glands of mice treated for 1 wk with JQ1 (gray background) or DAC, + progesterone. Dead cells were determined via propidium iodide staining; biological replicates: n ≥7 (JQ1) or n ≥4 (DAC). Statistical significance was tested for using one-way ANOVA followed by Dunnett’s multiple comparisons test; no comparisons were found to be statistically significant. (G) Bar charts show relative frequency of early- and late-apoptotic mammary cells after treatment with 0.5 mg/kg DAC, three weekly doses for 4 wk, determined via annexin V and propidium iodide staining. Biological replicates, n = 5. Statistical significance was tested using two-way ANOVA followed by Sidak’s multiple comparison test; no comparisons were found to be statistically significant. *, P ≤ 0.05; **, P ≤ 0.01.

    Techniques Used: In Vivo, Flow Cytometry, Cytometry, Purification, Mouse Assay, Staining, Marker

    32) Product Images from "Functionally distinct disease-associated fibroblast subsets in rheumatoid arthritis"

    Article Title: Functionally distinct disease-associated fibroblast subsets in rheumatoid arthritis

    Journal: Nature Communications

    doi: 10.1038/s41467-018-02892-y

    Anatomical localization and proportions of fibroblast subsets. a Anatomical localization of fibroblast subsets and leukocytes in RA and OA synovial tissue. Hoechst 33258: white, CD45: cyan, PDPN: blue, CD34: green, THY1: red. Small arrowheads: CD34 – THY1 – fibroblasts. Big arrowheads: CD34 – THY1 + fibroblasts. Arrows: CD34 + fibroblasts. Scale = 100 µm. b Expression of fibroblast subset markers in RA and OA synovial tissue. Hoechst 33258: white, CDH11: cyan, PDPN: blue, CD34: green, THY1: red. c Proportions of fibroblast subsets in synovial tissue in OA ( n = 26) and RA ( n = 16) evaluated by flow cytometry. P values from the Wilcoxon's rank-sum test. d Proportions of CDH11 + cells in CD34 – THY1 – fibroblasts, CD34 – THY1 + fibroblasts, and CD34 + fibroblasts. L lining area, S sublining area
    Figure Legend Snippet: Anatomical localization and proportions of fibroblast subsets. a Anatomical localization of fibroblast subsets and leukocytes in RA and OA synovial tissue. Hoechst 33258: white, CD45: cyan, PDPN: blue, CD34: green, THY1: red. Small arrowheads: CD34 – THY1 – fibroblasts. Big arrowheads: CD34 – THY1 + fibroblasts. Arrows: CD34 + fibroblasts. Scale = 100 µm. b Expression of fibroblast subset markers in RA and OA synovial tissue. Hoechst 33258: white, CDH11: cyan, PDPN: blue, CD34: green, THY1: red. c Proportions of fibroblast subsets in synovial tissue in OA ( n = 26) and RA ( n = 16) evaluated by flow cytometry. P values from the Wilcoxon's rank-sum test. d Proportions of CDH11 + cells in CD34 – THY1 – fibroblasts, CD34 – THY1 + fibroblasts, and CD34 + fibroblasts. L lining area, S sublining area

    Techniques Used: Expressing, Flow Cytometry, Cytometry

    33) Product Images from "Protection against Dengue Virus Infection in Mice by Administration of Antibodies against Modified Nonstructural Protein 1"

    Article Title: Protection against Dengue Virus Infection in Mice by Administration of Antibodies against Modified Nonstructural Protein 1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0092495

    Anti-ΔC NS1 and anti-DJ NS1 Abs show lower binding activity to human endothelial cells and platelets than anti-full-length DENV NS1 Abs. (A) The C-terminal region of DENV NS1 protein from a.a. 271-352 was deleted to generate ΔC NS1 protein. The DJ NS1 protein consists of the N-terminus of DENV NS1 (a.a. 1-270) and the C-terminus of JEV NS1 (a.a. 271-352). Polyclonal Abs against DENV NS1, ΔC NS1, or DJ NS1 were generated in mice and purified on protein G columns. Formaldehyde-fixed human endothelial cells (B) and platelets (C) were incubated with control IgG, anti-DENV NS1, anti-ΔC NS1 or anti-DJ NS1 Abs, followed by Alexa 488-conjucated anti-mouse IgG and analyzed by flow cytometry. The averages of triplicate cultures are shown. *  P
    Figure Legend Snippet: Anti-ΔC NS1 and anti-DJ NS1 Abs show lower binding activity to human endothelial cells and platelets than anti-full-length DENV NS1 Abs. (A) The C-terminal region of DENV NS1 protein from a.a. 271-352 was deleted to generate ΔC NS1 protein. The DJ NS1 protein consists of the N-terminus of DENV NS1 (a.a. 1-270) and the C-terminus of JEV NS1 (a.a. 271-352). Polyclonal Abs against DENV NS1, ΔC NS1, or DJ NS1 were generated in mice and purified on protein G columns. Formaldehyde-fixed human endothelial cells (B) and platelets (C) were incubated with control IgG, anti-DENV NS1, anti-ΔC NS1 or anti-DJ NS1 Abs, followed by Alexa 488-conjucated anti-mouse IgG and analyzed by flow cytometry. The averages of triplicate cultures are shown. * P

    Techniques Used: Binding Assay, Activity Assay, Generated, Mouse Assay, Purification, Incubation, Flow Cytometry, Cytometry

    34) Product Images from "Maturation of Lymph Node Fibroblastic Reticular Cells from Myofibroblastic Precursors Is Critical for Antiviral Immunity"

    Article Title: Maturation of Lymph Node Fibroblastic Reticular Cells from Myofibroblastic Precursors Is Critical for Antiviral Immunity

    Journal: Immunity

    doi: 10.1016/j.immuni.2013.03.012

    FRC-Specific LTβR Ablation Alters LN Stromal Cell Composition and Phenotype (A) Frequency of the major stromal cell populations (PDPN − CD31 − DN cells, PDPN + CD31 − [FRC], PDPN + CD31 + [LEC]; PDPN − CD31 + [BEC]) in inguinal LNs from Ccl19-cre × Ltbr fl/fl and Ltbr +/+ mice as determined by flow cytometry; values represent mean ± SEM (n = 9 mice from three independent experiments). (B) Representative LTβR expression patterns on PDPN + CD31 − (FRC) stromal cells of Ccl19-cre × Ltbr fl/fl (red) and Ltbr +/+ (black) mice determined by flow cytometry; staining with isotype control antibody in gray. (C) Summary of LTβR expression displayed as mean fluorescent intensity (MFI), mean ± SEM (n = 4 mice from two independent experiments). (D–G) Confocal microscopic analysis of inguinal LN stromal cells from 6-week-old Ccl19-cre × Ltbr fl/fl and Ltbr +/+ controls. (D) T cell zone reticular network revealed by ERTR-7 and PDPN staining, merged channels in left panel; scale bar represents 20 μm. (E) High-resolution analysis of T cell zone conduit with collagen-1 bundles ensheathed by ERTR-7; scale bar represents 2 μm. (F) Drainage of 40 kDa FITC dextran through LN conduits 2 min after subcutaneous injection. Left panel shows magnified FITC staining in T cell zone; scale bar represents 100/40 μm. (G) Vascular (HEV, high endothelial venule) and T cell zone stromal cell-associated CCL21 expression, merged channels in left panel, CD31 and CCL21 merge in right panel; scale bar represents 20 μm. (H–K) Inguinal LNs from 6-week-old Ccl19-cre × Ltbr fl/fl and Ltbr +/+ controls were analyzed by quantitative RT-PCR for the expression of Ccl21 (H), Ccl19 (I), Il7 (J), and Cxcl13 (K). Values indicate mean ± SEM from two individual LNs from three mice analyzed in two independent experiments.
    Figure Legend Snippet: FRC-Specific LTβR Ablation Alters LN Stromal Cell Composition and Phenotype (A) Frequency of the major stromal cell populations (PDPN − CD31 − DN cells, PDPN + CD31 − [FRC], PDPN + CD31 + [LEC]; PDPN − CD31 + [BEC]) in inguinal LNs from Ccl19-cre × Ltbr fl/fl and Ltbr +/+ mice as determined by flow cytometry; values represent mean ± SEM (n = 9 mice from three independent experiments). (B) Representative LTβR expression patterns on PDPN + CD31 − (FRC) stromal cells of Ccl19-cre × Ltbr fl/fl (red) and Ltbr +/+ (black) mice determined by flow cytometry; staining with isotype control antibody in gray. (C) Summary of LTβR expression displayed as mean fluorescent intensity (MFI), mean ± SEM (n = 4 mice from two independent experiments). (D–G) Confocal microscopic analysis of inguinal LN stromal cells from 6-week-old Ccl19-cre × Ltbr fl/fl and Ltbr +/+ controls. (D) T cell zone reticular network revealed by ERTR-7 and PDPN staining, merged channels in left panel; scale bar represents 20 μm. (E) High-resolution analysis of T cell zone conduit with collagen-1 bundles ensheathed by ERTR-7; scale bar represents 2 μm. (F) Drainage of 40 kDa FITC dextran through LN conduits 2 min after subcutaneous injection. Left panel shows magnified FITC staining in T cell zone; scale bar represents 100/40 μm. (G) Vascular (HEV, high endothelial venule) and T cell zone stromal cell-associated CCL21 expression, merged channels in left panel, CD31 and CCL21 merge in right panel; scale bar represents 20 μm. (H–K) Inguinal LNs from 6-week-old Ccl19-cre × Ltbr fl/fl and Ltbr +/+ controls were analyzed by quantitative RT-PCR for the expression of Ccl21 (H), Ccl19 (I), Il7 (J), and Cxcl13 (K). Values indicate mean ± SEM from two individual LNs from three mice analyzed in two independent experiments.

    Techniques Used: Mouse Assay, Flow Cytometry, Expressing, Staining, Injection, Quantitative RT-PCR

    LTβR-Dependent FRC Maturation (A) LN stromal cells from 6-week-old Ccl19-cre × R26-eyfp mice with wild-type ( +/+ , WT) and heterozygously ( +/fl ) or homozygously floxed ( fl/fl ) Ltbr loci were assessed by flow cytometry for EYFP expression by using back gating. Representative dot plot analysis with quadstat values of CD31 and PDPN expression is shown. (B) EYFP expression in FRCs and DN cells in the indicated Ltbr genotype of Ccl19-cre × R26-eyfp mice; mean ± SEM (n = 3 mice from two independent experiments). (C) Representative analysis of ICAM-1 and VCAM-1 expression on Ltbr +/+ DN cells from Ccl19-cre × R26-eyfp mice (black), EYFP + Ltbr +/+ FRCs of Ccl19-cre × R26-eyfp mice (red), and EYFP + cells of Ccl19-cre × R26-eyfp x Ltbr fl/fl mice (blue). (D) Inguinal LNs from 6-week-old Ccl19-cre × Ltbr fl/fl and Ltbr +/+ controls were analyzed by quantitative RT-PCR for the expression of Acta2 (SMA), Cnn1 (Calponin-1), Pdgfrb , Pdgfra , and Cspg4 (NG2). Values indicate mean ± SEM from two individual LNs from > 3 mice analyzed in two independent experiments. (E–I) Confocal microscopic analysis of inguinal LN stromal cells from 6-week-old Ccl19-cre × R26-eyfp x Ltbr fl/fl mice and Ltbr +/+ Ccl19-cre × R26-eyfp controls. Reconstruction of perivascular (E) and T cell zonal (F) stromal cell network by analyzing CD31, SMA, and EYFP expression is shown. Arrowheads indicate perivascular EYFP expression, arrows indicate SMA + EYFP + cells in orthogonal sections, and scale bars represent 10 μm. (G) Analysis of perivascular and T cell zonal expression of EYFP and PDGFRβ; scale bar represents 20 μm, all panels show merged channels. Reconstruction of perivascular and network-forming cells expressing CNN1 (H) and NG2 (I) and EYFP, merged channels in left panels; scale bars represent 30 μm; representative data out of three independent experiments. See also Figure S3 .
    Figure Legend Snippet: LTβR-Dependent FRC Maturation (A) LN stromal cells from 6-week-old Ccl19-cre × R26-eyfp mice with wild-type ( +/+ , WT) and heterozygously ( +/fl ) or homozygously floxed ( fl/fl ) Ltbr loci were assessed by flow cytometry for EYFP expression by using back gating. Representative dot plot analysis with quadstat values of CD31 and PDPN expression is shown. (B) EYFP expression in FRCs and DN cells in the indicated Ltbr genotype of Ccl19-cre × R26-eyfp mice; mean ± SEM (n = 3 mice from two independent experiments). (C) Representative analysis of ICAM-1 and VCAM-1 expression on Ltbr +/+ DN cells from Ccl19-cre × R26-eyfp mice (black), EYFP + Ltbr +/+ FRCs of Ccl19-cre × R26-eyfp mice (red), and EYFP + cells of Ccl19-cre × R26-eyfp x Ltbr fl/fl mice (blue). (D) Inguinal LNs from 6-week-old Ccl19-cre × Ltbr fl/fl and Ltbr +/+ controls were analyzed by quantitative RT-PCR for the expression of Acta2 (SMA), Cnn1 (Calponin-1), Pdgfrb , Pdgfra , and Cspg4 (NG2). Values indicate mean ± SEM from two individual LNs from > 3 mice analyzed in two independent experiments. (E–I) Confocal microscopic analysis of inguinal LN stromal cells from 6-week-old Ccl19-cre × R26-eyfp x Ltbr fl/fl mice and Ltbr +/+ Ccl19-cre × R26-eyfp controls. Reconstruction of perivascular (E) and T cell zonal (F) stromal cell network by analyzing CD31, SMA, and EYFP expression is shown. Arrowheads indicate perivascular EYFP expression, arrows indicate SMA + EYFP + cells in orthogonal sections, and scale bars represent 10 μm. (G) Analysis of perivascular and T cell zonal expression of EYFP and PDGFRβ; scale bar represents 20 μm, all panels show merged channels. Reconstruction of perivascular and network-forming cells expressing CNN1 (H) and NG2 (I) and EYFP, merged channels in left panels; scale bars represent 30 μm; representative data out of three independent experiments. See also Figure S3 .

    Techniques Used: Mouse Assay, Flow Cytometry, Expressing, Quantitative RT-PCR

    Ccl19-cre Transgene Expression in LN Stromal Cells (A) LN cell suspensions from 6-week-old Ccl19-cre × R26-eyfp mice were depleted of CD45 + cells, and EYFP expression was determined by flow cytometry. (B) Representative dot plot analysis with quadstat values of CD31 and PDPN expression in CD45 − inguinal LN stromal cells. (C) Dot plot analysis including quadstat values of CD31 and PDPN expression in CD45 − EYFP + LN stromal cells. (D) EYFP expression in CD45 − LN stromal cell populations (PDPN + CD31 − fibroblastic reticular cells [FRC]; PDPN − CD31 − double-negative [DN] cells; PDPN + CD31 + lymphatic endothelial cells [LECs]; PDPN − CD31 + blood endothelial cells [BEC]). Inguinal LNs from individual mice were pooled, mean values ± SEM from 15 mice analyzed in three independent experiments. (E) Confocal microscopic analysis of an Ccl19-cre × R26-eyfp inguinal LN section by using antibodies against the indicated markers, merged channels in left panel; scale bar represents 200 μm. (F) 3D reconstruction of T cell zone FRC network, merged channels in left panel, scale bar = 10 μm. (G) EYFP expression in perivascular FRCs (arrow); scale bar represents 20 μm. (H and I) Expression of the homeostatic chemokines CCL21 (H, arrows) and CCL19 (I, arrows) in EYFP + FRCs in T cell zones of Ccl19-cre × R26-eyfp inguinal LNs; scale bar represents 20 μm. See also Figure S1 .
    Figure Legend Snippet: Ccl19-cre Transgene Expression in LN Stromal Cells (A) LN cell suspensions from 6-week-old Ccl19-cre × R26-eyfp mice were depleted of CD45 + cells, and EYFP expression was determined by flow cytometry. (B) Representative dot plot analysis with quadstat values of CD31 and PDPN expression in CD45 − inguinal LN stromal cells. (C) Dot plot analysis including quadstat values of CD31 and PDPN expression in CD45 − EYFP + LN stromal cells. (D) EYFP expression in CD45 − LN stromal cell populations (PDPN + CD31 − fibroblastic reticular cells [FRC]; PDPN − CD31 − double-negative [DN] cells; PDPN + CD31 + lymphatic endothelial cells [LECs]; PDPN − CD31 + blood endothelial cells [BEC]). Inguinal LNs from individual mice were pooled, mean values ± SEM from 15 mice analyzed in three independent experiments. (E) Confocal microscopic analysis of an Ccl19-cre × R26-eyfp inguinal LN section by using antibodies against the indicated markers, merged channels in left panel; scale bar represents 200 μm. (F) 3D reconstruction of T cell zone FRC network, merged channels in left panel, scale bar = 10 μm. (G) EYFP expression in perivascular FRCs (arrow); scale bar represents 20 μm. (H and I) Expression of the homeostatic chemokines CCL21 (H, arrows) and CCL19 (I, arrows) in EYFP + FRCs in T cell zones of Ccl19-cre × R26-eyfp inguinal LNs; scale bar represents 20 μm. See also Figure S1 .

    Techniques Used: Expressing, Mouse Assay, Flow Cytometry

    35) Product Images from "Influenza Virus Infects Epithelial Stem/Progenitor Cells of the Distal Lung: Impact on Fgfr2b-Driven Epithelial Repair"

    Article Title: Influenza Virus Infects Epithelial Stem/Progenitor Cells of the Distal Lung: Impact on Fgfr2b-Driven Epithelial Repair

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005544

    EpiSPC are resistant to apoptosis and show a high proliferative response after PR/8 infection which is mediated by Fgf10/Fgfr2b signaling. (A) Proliferation rates of the given epithelial cell subsets was analysed in PR/8 infected wt mice by FACS quantification of Ki67 + cells at the indicated time points pi. (B) Apoptosis of each EpCam + subset was quantified by FACS (Annexin V + proportions) at d7 post PR/8 infection and of non-infected wt mice. (C) Expression of Fgfr2b on EpiSPC at the given time points post PR/8 or mock infection was quantified by FACS and is given as MFI (median fluorescence intensity) of Fgfr2b ab minus MFI of matched isotype control. The proliferative response of the EpCam + cell subsets was quantified by FACS at d7 pi in Rosa26 rtTA/+ ;tet(O)sFgfr2b/+ (D) Rosa26 rtTA/+ ;tet(O)Fgf10/+ mice (E) and Fgf7 -/- mice (F) compared to non-dox-induced or wt littermates. Bar graphs represent means ± SD of n = 4–6 independent experiments; * p
    Figure Legend Snippet: EpiSPC are resistant to apoptosis and show a high proliferative response after PR/8 infection which is mediated by Fgf10/Fgfr2b signaling. (A) Proliferation rates of the given epithelial cell subsets was analysed in PR/8 infected wt mice by FACS quantification of Ki67 + cells at the indicated time points pi. (B) Apoptosis of each EpCam + subset was quantified by FACS (Annexin V + proportions) at d7 post PR/8 infection and of non-infected wt mice. (C) Expression of Fgfr2b on EpiSPC at the given time points post PR/8 or mock infection was quantified by FACS and is given as MFI (median fluorescence intensity) of Fgfr2b ab minus MFI of matched isotype control. The proliferative response of the EpCam + cell subsets was quantified by FACS at d7 pi in Rosa26 rtTA/+ ;tet(O)sFgfr2b/+ (D) Rosa26 rtTA/+ ;tet(O)Fgf10/+ mice (E) and Fgf7 -/- mice (F) compared to non-dox-induced or wt littermates. Bar graphs represent means ± SD of n = 4–6 independent experiments; * p

    Techniques Used: Infection, Mouse Assay, FACS, Expressing, Fluorescence

    Therapeutic treatment with recombinant Fgf10 improves influenza virus-induced lung injury and improves re-epithelialization and barrier repair. Wt mice were infected with PR/8 and treated with a single dose of either 5μg recombinant Fgf10 (rFgf10) or diluent (PBS -/- ) at d6 pi. (A) The proliferative response of EpCam + epithelial cell subsets was determined by flow cytometry at d7 pi. (B) Lung sections were stained with hematoxylin-eosin at d10 and d21 pi. Arrows depict non-epithelialized alveolar tissue; arrowheads depict areas of ongoing re-epithelialization. (C) Immunofluorescence staining of lung sections for E-cadherin (green), Ki67 (red), and Dapi (blue) at d21 pi. The top row shows lung tissue from mock-infected, untreated mice at d21. (D) Quantification of total lung epithelial cells (EpCam + ) in lung homogenates at d14 pi. (E) Lung sections were stained for krt5 (green) and Dapi (blue) at d21 pi. (F) Lung barrier function was analysed by quantification of alveolar leakage of FITC-labeled albumin at d14 pi. Values are given in arbitrary units (AU) and represent ratios of FITC fluorescence in BALF and serum. (G) Survival of n = 8 mice per treatment group was analysed until d21 pi. Bar graphs represent means ± SD of n = 5–6 independent experiments; * p
    Figure Legend Snippet: Therapeutic treatment with recombinant Fgf10 improves influenza virus-induced lung injury and improves re-epithelialization and barrier repair. Wt mice were infected with PR/8 and treated with a single dose of either 5μg recombinant Fgf10 (rFgf10) or diluent (PBS -/- ) at d6 pi. (A) The proliferative response of EpCam + epithelial cell subsets was determined by flow cytometry at d7 pi. (B) Lung sections were stained with hematoxylin-eosin at d10 and d21 pi. Arrows depict non-epithelialized alveolar tissue; arrowheads depict areas of ongoing re-epithelialization. (C) Immunofluorescence staining of lung sections for E-cadherin (green), Ki67 (red), and Dapi (blue) at d21 pi. The top row shows lung tissue from mock-infected, untreated mice at d21. (D) Quantification of total lung epithelial cells (EpCam + ) in lung homogenates at d14 pi. (E) Lung sections were stained for krt5 (green) and Dapi (blue) at d21 pi. (F) Lung barrier function was analysed by quantification of alveolar leakage of FITC-labeled albumin at d14 pi. Values are given in arbitrary units (AU) and represent ratios of FITC fluorescence in BALF and serum. (G) Survival of n = 8 mice per treatment group was analysed until d21 pi. Bar graphs represent means ± SD of n = 5–6 independent experiments; * p

    Techniques Used: Recombinant, Mouse Assay, Infection, Flow Cytometry, Cytometry, Staining, Immunofluorescence, Labeling, Fluorescence

    36) Product Images from "The Lymphatic Endothelial mCLCA1 Antibody Induces Proliferation and Growth of Lymph Node Lymphatic Sinuses"

    Article Title: The Lymphatic Endothelial mCLCA1 Antibody Induces Proliferation and Growth of Lymph Node Lymphatic Sinuses

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0156079

    10.1.1 Ab induces coordinate accumulation of lymphatic endothelial and stromal cells. Pooled LNs were enzymatically digested and stained with CD45, CD31, and Podoplanin antibodies for flow cytometry analysis. Viable cells were gated as CD45- to detect all stroma and then further gated to identify LEC (CD31+ Podoplanin+), FRC (CD31- Podoplanin+), BEC (CD31+ Podoplanin-), and DN stromal cells (CD31- Podoplanin-). A). 10.1.1 Ab-injected mice display an increased percentage of increased CD45- cells. B). Populations are shown as a percent of CD45- cells. All cell types remain proportional in the 10.1.1 Ab-injected mice indicating a coordinate expansion of LN stromal cell populations in response to 10.1.1 Ab treatment. Significance was determined using a Mann Whitney U test for unpaired samples. n = 6, p
    Figure Legend Snippet: 10.1.1 Ab induces coordinate accumulation of lymphatic endothelial and stromal cells. Pooled LNs were enzymatically digested and stained with CD45, CD31, and Podoplanin antibodies for flow cytometry analysis. Viable cells were gated as CD45- to detect all stroma and then further gated to identify LEC (CD31+ Podoplanin+), FRC (CD31- Podoplanin+), BEC (CD31+ Podoplanin-), and DN stromal cells (CD31- Podoplanin-). A). 10.1.1 Ab-injected mice display an increased percentage of increased CD45- cells. B). Populations are shown as a percent of CD45- cells. All cell types remain proportional in the 10.1.1 Ab-injected mice indicating a coordinate expansion of LN stromal cell populations in response to 10.1.1 Ab treatment. Significance was determined using a Mann Whitney U test for unpaired samples. n = 6, p

    Techniques Used: Staining, Flow Cytometry, Cytometry, Injection, Mouse Assay, MANN-WHITNEY

    10.1.1 Ab-induced lymphangiogenesis is not due to enlargement of LECs. A). Pooled LNs were enzymatically digested and analyzed by flow cytometry. No difference in forward scatter profiles was identified between control Hamster IgG- and 10.1.1 Ab-injected mice, indicating no difference in cell size between the two treatment groups. Overlay of forward scatter profile histograms gated on viable CD45- CD31+ Podoplanin+ LECs from representative samples of Hamster IgG- and 10.1.1 Ab-injected mice are shown. B). Bar graph represents the average arithmetic mean and standard error from 2 independent experiments. n = 6.
    Figure Legend Snippet: 10.1.1 Ab-induced lymphangiogenesis is not due to enlargement of LECs. A). Pooled LNs were enzymatically digested and analyzed by flow cytometry. No difference in forward scatter profiles was identified between control Hamster IgG- and 10.1.1 Ab-injected mice, indicating no difference in cell size between the two treatment groups. Overlay of forward scatter profile histograms gated on viable CD45- CD31+ Podoplanin+ LECs from representative samples of Hamster IgG- and 10.1.1 Ab-injected mice are shown. B). Bar graph represents the average arithmetic mean and standard error from 2 independent experiments. n = 6.

    Techniques Used: Flow Cytometry, Cytometry, Injection, Mouse Assay

    37) Product Images from "MyD88-dependent Dendritic and Epithelial Cell Crosstalk Orchestrates Immune Responses to Allergens"

    Article Title: MyD88-dependent Dendritic and Epithelial Cell Crosstalk Orchestrates Immune Responses to Allergens

    Journal: Mucosal immunology

    doi: 10.1038/mi.2017.84

    Differential control of Th2 and Th17 cytokine production by EC and DC expression of Myd88 ( a–b ) Cytokines in mLNs after a single sensitization ( a ) or two sensitizations ( b ) with 100 μg OVA and 1.25 μg FLA. ( c ) T cell proliferation and cytokine production in co-cultures of OT-II T cells mixed with the indicated APC(s) sorted from lungs of mice 6h after sensitization with OVA/FLA. ( d ) Intracellular staining for IL-17 and type 2 cytokines in CD4 + T cells of mice sensitized with OVA/FLA op on days 0 and 7 and harvested on day 10. Lung cells were stimulated with PMA and ionomycin prior to staining. Data shown are ( a ) representative of 2 experiments with 8–10 mice per group; ( b ) from a single experiment, representative of two, 4–5 mice per group; ( c ) representative of a single experiment performed with duplicate samples; and ( d ) from a single experiment with 3–4 mice per genotype. * p
    Figure Legend Snippet: Differential control of Th2 and Th17 cytokine production by EC and DC expression of Myd88 ( a–b ) Cytokines in mLNs after a single sensitization ( a ) or two sensitizations ( b ) with 100 μg OVA and 1.25 μg FLA. ( c ) T cell proliferation and cytokine production in co-cultures of OT-II T cells mixed with the indicated APC(s) sorted from lungs of mice 6h after sensitization with OVA/FLA. ( d ) Intracellular staining for IL-17 and type 2 cytokines in CD4 + T cells of mice sensitized with OVA/FLA op on days 0 and 7 and harvested on day 10. Lung cells were stimulated with PMA and ionomycin prior to staining. Data shown are ( a ) representative of 2 experiments with 8–10 mice per group; ( b ) from a single experiment, representative of two, 4–5 mice per group; ( c ) representative of a single experiment performed with duplicate samples; and ( d ) from a single experiment with 3–4 mice per genotype. * p

    Techniques Used: Expressing, Mouse Assay, Staining

    38) Product Images from "Pain-related behaviors and neurochemical alterations in mice expressing sickle hemoglobin: modulation by cannabinoids"

    Article Title: Pain-related behaviors and neurochemical alterations in mice expressing sickle hemoglobin: modulation by cannabinoids

    Journal: Blood

    doi: 10.1182/blood-2010-01-260372

    A montage of overlapping fields of z-stack images (each 2.5-μm-thick) of 80- to 100-μm-thick sections of skin showing the expression of mediators of pain in skin . HbA-BERK (A), hBERK1 (C), and BERK (E). Image shows CD31 + blood vessels (pseudo-colored green), SP immunoreactivity (pseudo-colored red), and CGRP immunoreactivity (pseudo-colored blue). Magenta color in panels C and E is indicative of colocalization of CGRP (blue) and SP (red). The inset in panel E shows localized, dense immunoreactivity for SP in BERK mice. Bar represents 100 μm. ep indicates epidermis. (B,D) Coexpression of MOR and CD31 + on blood vessels in HbA-BERK (B) and hBERK1 (D) mice. Image shows CD31 + blood vessels (pseudo-colored red), MOR immunoreactivity (pseudo-colored green), and 4,6-diamidino-2-phenylindole + nuclei (pseudo-colored blue). Yellow represents coexpression of MOR on blood vessels. Original magnification ×150; scale bar represents 250 μm. Arrows indicate MOR expression in the epidermis and vasculature. ep indicates epidermis. Each figure is representative of images from skins of 3 different mice. Image acquisition information: FluoView FV1000 Laser Scanning Confocal BX61 Microscope (Olympus), 20×/0.70 oil objective lens, In-built image acquisition system, Adobe Photoshop (panels A,C,E); Olympus IX70 microscope, 15×/0.45 objective lens, DP70 digital camera and DP70 Manager software (Olympus), Adobe Photoshop (panels B,D).
    Figure Legend Snippet: A montage of overlapping fields of z-stack images (each 2.5-μm-thick) of 80- to 100-μm-thick sections of skin showing the expression of mediators of pain in skin . HbA-BERK (A), hBERK1 (C), and BERK (E). Image shows CD31 + blood vessels (pseudo-colored green), SP immunoreactivity (pseudo-colored red), and CGRP immunoreactivity (pseudo-colored blue). Magenta color in panels C and E is indicative of colocalization of CGRP (blue) and SP (red). The inset in panel E shows localized, dense immunoreactivity for SP in BERK mice. Bar represents 100 μm. ep indicates epidermis. (B,D) Coexpression of MOR and CD31 + on blood vessels in HbA-BERK (B) and hBERK1 (D) mice. Image shows CD31 + blood vessels (pseudo-colored red), MOR immunoreactivity (pseudo-colored green), and 4,6-diamidino-2-phenylindole + nuclei (pseudo-colored blue). Yellow represents coexpression of MOR on blood vessels. Original magnification ×150; scale bar represents 250 μm. Arrows indicate MOR expression in the epidermis and vasculature. ep indicates epidermis. Each figure is representative of images from skins of 3 different mice. Image acquisition information: FluoView FV1000 Laser Scanning Confocal BX61 Microscope (Olympus), 20×/0.70 oil objective lens, In-built image acquisition system, Adobe Photoshop (panels A,C,E); Olympus IX70 microscope, 15×/0.45 objective lens, DP70 digital camera and DP70 Manager software (Olympus), Adobe Photoshop (panels B,D).

    Techniques Used: Expressing, Mouse Assay, Microscopy, Software

    39) Product Images from "Human Term Placenta as a Source of Hematopoietic Cells"

    Article Title: Human Term Placenta as a Source of Hematopoietic Cells

    Journal: Experimental Biology and Medicine (Maywood, N.j.)

    doi: 10.3181/0809-BC-262

    Hematopoietic cells in human placenta. Multiple clusters of CD34+cells, not associated with fetal or maternal circulation, are present in placental tissue. Significant numbers of CD34 cells are also positive for hematopoietic precursor markers such as CD90, CD38 and CD133. Paraffin sections of human placenta stained for CD34, CD90, CD38, and CD133 are shown. A1 : CD90 (green), A2 : CD34 (red), A3 : merged image of A1 and A2 with nuclear co-staining (DAPI blue), size indication bar is 10 µm. The white arrow points to CD34+CD90+cells. B1 : CD38 (green), B2 : CD34 (red), B3 : merged image of B1 and B2 with nuclear co-staining (DAPI blue), size indication bar is 100 µm. The white arrows point to CD34+ CD38+ cells. The green arrow points at a CD34+ capillary. C1 : CD133 (green), C2 : CD34 (red), C3 : merged image, showing staining for CD34 (red), CD133 (green), and nuclei (DAPI blue), size indication bar is 20 µm. The white arrows point to CD34+CD133+cells not associated with structures of the circulation (capillary walls). The green arrow points at the CD34+CD133+capillary. D : Negative control staining. D1 : merged image of staining with isotype anti-mouse antibody and isotype anti-rabbit antibody, secondary FITC (green) anti-mouse and Alexa Fluor 633 anti-rabbit antibody (red), co-stained for nuclei with DAPI (blue). No signals from cell markers other than nuclei are present. D2 : Merged image of staining with mouse anti-CD34 antibody and isotype anti-rabbit antibody, secondary FITC anti-mouse (green) and Alexa Fluor 633 anti-rabbit (red) antibody, co-stained for nuclei with DAPI (blue). Only signals from the CD34 cell marker and nuclei are present. D3 : Isotype anti-mouse antibody and anti-CD133 rabbit antibody, secondary FITC anti-mouse and Alexa Fluor 633 anti-rabbit antibody, co-staining DAPI (blue), merged image. Only a signal for CD133+cells is observed, no signals from CD34 cell markers are present. The size indication bar is 50 µm.
    Figure Legend Snippet: Hematopoietic cells in human placenta. Multiple clusters of CD34+cells, not associated with fetal or maternal circulation, are present in placental tissue. Significant numbers of CD34 cells are also positive for hematopoietic precursor markers such as CD90, CD38 and CD133. Paraffin sections of human placenta stained for CD34, CD90, CD38, and CD133 are shown. A1 : CD90 (green), A2 : CD34 (red), A3 : merged image of A1 and A2 with nuclear co-staining (DAPI blue), size indication bar is 10 µm. The white arrow points to CD34+CD90+cells. B1 : CD38 (green), B2 : CD34 (red), B3 : merged image of B1 and B2 with nuclear co-staining (DAPI blue), size indication bar is 100 µm. The white arrows point to CD34+ CD38+ cells. The green arrow points at a CD34+ capillary. C1 : CD133 (green), C2 : CD34 (red), C3 : merged image, showing staining for CD34 (red), CD133 (green), and nuclei (DAPI blue), size indication bar is 20 µm. The white arrows point to CD34+CD133+cells not associated with structures of the circulation (capillary walls). The green arrow points at the CD34+CD133+capillary. D : Negative control staining. D1 : merged image of staining with isotype anti-mouse antibody and isotype anti-rabbit antibody, secondary FITC (green) anti-mouse and Alexa Fluor 633 anti-rabbit antibody (red), co-stained for nuclei with DAPI (blue). No signals from cell markers other than nuclei are present. D2 : Merged image of staining with mouse anti-CD34 antibody and isotype anti-rabbit antibody, secondary FITC anti-mouse (green) and Alexa Fluor 633 anti-rabbit (red) antibody, co-stained for nuclei with DAPI (blue). Only signals from the CD34 cell marker and nuclei are present. D3 : Isotype anti-mouse antibody and anti-CD133 rabbit antibody, secondary FITC anti-mouse and Alexa Fluor 633 anti-rabbit antibody, co-staining DAPI (blue), merged image. Only a signal for CD133+cells is observed, no signals from CD34 cell markers are present. The size indication bar is 50 µm.

    Techniques Used: Staining, Negative Control, Marker

    40) Product Images from "Low-temperature culturing improves survival rate of tissue-engineered cardiac cell sheets"

    Article Title: Low-temperature culturing improves survival rate of tissue-engineered cardiac cell sheets

    Journal: Biochemistry and Biophysics Reports

    doi: 10.1016/j.bbrep.2018.04.001

    Implantation of quintuple-layered cell sheets into nude rats. (A) Schematic illustration of the implantation of quintuple-layered cell sheets into nude rats. (B) The top view microphotograph of a CD31-stained quintuple-layered cell sheet, which was cultured at 30 °C for 7 days to produce a vascular network (scale bar, 100 µm). (C) The top view microphotograph of a CD31-stained, freshly prepared quintuple-layered cell sheet. The former cell sheet, which was obtained by cultivation at 30 °C, shows dense vascular networks (scale bar, 100 µm). (D) Schematic illustration shows the transplanted site of a quintuple-layered cell sheet. (E) Macro-photograph of a transplanted cell sheet on the tissue of a rat (scale bar, 10 mm). At 2 weeks after transplantation, the transplanted cell sheet was removed from the nude rat. The specimens were stained with hematoxylin–eosin for histological analysis (F, G) (scale bars, 50 µm). The low-temperature-treated quintuple-layered cell sheet (F) was found to be engrafted more clearly than the standard cell sheet (G), and the remaining cardiomyocytes were confirmed by troponin T staining (H, I) (scale bars, 50 µm).
    Figure Legend Snippet: Implantation of quintuple-layered cell sheets into nude rats. (A) Schematic illustration of the implantation of quintuple-layered cell sheets into nude rats. (B) The top view microphotograph of a CD31-stained quintuple-layered cell sheet, which was cultured at 30 °C for 7 days to produce a vascular network (scale bar, 100 µm). (C) The top view microphotograph of a CD31-stained, freshly prepared quintuple-layered cell sheet. The former cell sheet, which was obtained by cultivation at 30 °C, shows dense vascular networks (scale bar, 100 µm). (D) Schematic illustration shows the transplanted site of a quintuple-layered cell sheet. (E) Macro-photograph of a transplanted cell sheet on the tissue of a rat (scale bar, 10 mm). At 2 weeks after transplantation, the transplanted cell sheet was removed from the nude rat. The specimens were stained with hematoxylin–eosin for histological analysis (F, G) (scale bars, 50 µm). The low-temperature-treated quintuple-layered cell sheet (F) was found to be engrafted more clearly than the standard cell sheet (G), and the remaining cardiomyocytes were confirmed by troponin T staining (H, I) (scale bars, 50 µm).

    Techniques Used: Staining, Cell Culture, Transplantation Assay

    Related Articles

    Microscopy:

    Article Title: Transplantation of Mesenchymal Stem Cells for Acute Spinal Cord Injury in Rats: Comparative Study between Intralesional Injection and Scaffold Based Transplantation
    Article Snippet: .. Cells that were positive for these markers (green) were co-stained with PKH 26 and DAPI (4',6-Diamidino-2-Phenylindole; blue, Invitrogen, Eugene, OR, USA), and were counted using a confocal microscope. .. For quantitative analysis of the engraftment of transplanted MSCs according to each transplantation route, cells expressing co-staining of PKH 26 and DAPI were counted in six fields under high-power magnification (× 400).

    Article Title: Speckled-like Pattern in the Germinal Center (SLIP-GC), a Nuclear GTPase Expressed in Activation-induced Deaminase-expressing Lymphomas and Germinal Center B Cells *
    Article Snippet: .. The DNA was counterstained with DAPI, and slides were mounted in prolong antifade media (Molecular Probes), dried, and viewed under confocal microscope (LSM 510 mounted on an Axiovert 200 m microscope, Carl Zeiss, Inc.). .. 293 cells were grown on coverslips and transfected with the SLIP-GC-pEGFP-C3 vector as described above.

    Staining:

    Article Title: Schizophrenia patient-derived olfactory neurosphere-derived cells do not respond to extracellular reelin
    Article Snippet: .. Nuclei and cell cytoplasm were stained with DAPI (1:1,000; Life Technologies) and CellMask Deep Red Plasma Membrane Stain (1:5,000; Life Technologies), respectively. ..

    Article Title: Human Cytomegalovirus pUS24 Is a Virion Protein That Functions Very Early in the Replication Cycle
    Article Snippet: .. In some cases, the secondary antibody was supplemented with 1 μg/ml of DAPI (4′,6′-diamidino-2-phenylindole; Molecular Probes) to stain DNA. .. For colocalization studies, mouse monoclonal antibody against US24 and rabbit polyclonal antibody against pp65 (gift from Martin Schrader, University of Marburg) were used in the primary-antibody incubation.

    Article Title: Entry, Intracellular Survival, and Multinucleated-Giant-Cell-Forming Activity of Burkholderia pseudomallei in Human Primary Phagocytic and Nonphagocytic Cells
    Article Snippet: .. The cell mixture was infected with B. thailandensis wild-type and Δ tssK-5 strains at an MOI of 10 for approximately 22 h, fixed with 4% formaldehyde, and stained with wheat germ agglutinin Alexa Fluor 594 conjugate and DAPI (Invitrogen). .. The samples were mounted using ProLong Diamond (Invitrogen), and images were collected using the confocal mode of an inverted Zeiss LSM 710 NLO microscope equipped with a spectral detector, a Zeiss Plan-Apochromat 63×/1.40 oil DIC M27 objective and the Zeiss Zen 2011 software.

    Incubation:

    Article Title: The Nucleotide Excision Repair Pathway Protects Borrelia burgdorferi from Nitrosative Stress in Ixodes scapularis Ticks
    Article Snippet: .. Tick tissues were incubated in PBS in the presence of BSA (10 mg ml-1 ), and rabbit serum as a negative control or with a 1:100 dilution of a universal NOS antibody (Thermo Fisher) for 3 h. Tissues were then subjected to 3 × 0.5 ml washes in PBS, and then incubated with a 1:200 dilution of the Goat anti-Rabbit IgG secondary antibody Alexa Fluor 488 conjugate (Life Technologies) and a 1:400 dilution of DAPI (Thermo Fisher) for an additional 3 h at RT. .. Tissues were washed as described above, and then visualized by fluorescent microscopy using the Blue channel (Exλ = 355 nm, Emλ = 433 nm) and the Green channel (Exλ = 480 nm, Emλ = 517 nm) of the ZOE Flourescent Cell Imager (Bio-Rad).

    Article Title: A Fluorescent Cell-Based System for Imaging Zika Virus Infection in Real-Time
    Article Snippet: .. Following primary antibody incubation for 2 h, slides were washed 3× with PBS, and immunostained with conjugated AlexaFluor secondary antibodies (Life Technologies, Carlsbad, CA, USA, 1:500), along with DAPI (4′,6-diamidino-2-phenylindole) (Life Technologies, 1:500), and mounted with ProLong Gold (Invitrogen, Carlsbad, CA, USA). .. Confocal microscopy was performed on a Leica SP5 (Wetzlar, Germany) inverted confocal microscope using a 40×/1.25 oil objective using 405, 488, 561 and 633 laser lines at a 4× optical zoom with pinholes set to 1 Airy unit for each channel (Light Microscopy Core Facility, Duke University).

    Article Title: HUWE1 plays important role in mouse preimplantation embryo development and the dysregulation is associated with poor embryo development in humans
    Article Snippet: .. Embryos were washed five times in 1% BSA/PBS, after then incubated in 2 μg/ml DAPI for 8 min, washed once and mounted on glass slides with Gold anti-fade reagent (P36934, Life Technologies, USA). ..

    Negative Control:

    Article Title: The Nucleotide Excision Repair Pathway Protects Borrelia burgdorferi from Nitrosative Stress in Ixodes scapularis Ticks
    Article Snippet: .. Tick tissues were incubated in PBS in the presence of BSA (10 mg ml-1 ), and rabbit serum as a negative control or with a 1:100 dilution of a universal NOS antibody (Thermo Fisher) for 3 h. Tissues were then subjected to 3 × 0.5 ml washes in PBS, and then incubated with a 1:200 dilution of the Goat anti-Rabbit IgG secondary antibody Alexa Fluor 488 conjugate (Life Technologies) and a 1:400 dilution of DAPI (Thermo Fisher) for an additional 3 h at RT. .. Tissues were washed as described above, and then visualized by fluorescent microscopy using the Blue channel (Exλ = 355 nm, Emλ = 433 nm) and the Green channel (Exλ = 480 nm, Emλ = 517 nm) of the ZOE Flourescent Cell Imager (Bio-Rad).

    Infection:

    Article Title: Entry, Intracellular Survival, and Multinucleated-Giant-Cell-Forming Activity of Burkholderia pseudomallei in Human Primary Phagocytic and Nonphagocytic Cells
    Article Snippet: .. The cell mixture was infected with B. thailandensis wild-type and Δ tssK-5 strains at an MOI of 10 for approximately 22 h, fixed with 4% formaldehyde, and stained with wheat germ agglutinin Alexa Fluor 594 conjugate and DAPI (Invitrogen). .. The samples were mounted using ProLong Diamond (Invitrogen), and images were collected using the confocal mode of an inverted Zeiss LSM 710 NLO microscope equipped with a spectral detector, a Zeiss Plan-Apochromat 63×/1.40 oil DIC M27 objective and the Zeiss Zen 2011 software.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95
    Thermo Fisher cd31 monoclonal antibody
    Altered alveologenesis in PC-specific Yap1 and Wwtr1 mutant mice. a Three-dimensional reconstruction confocal images of Yap1 , Wwtr1 iPCKO and littermate control lungs stained for AQP5 (green), PDGFRβ (red), and PECAM1 (blue) at the indicated stages. Scale bar, 50 µm. b Quantitation of airspace volume in Yap1 , Wwtr1 iPCKO and control lung sections with three-dimensional reconstruction surface images. Data represents mean ± s.e.m. ( n = 4 mice; unpaired two tailed student t -test or Welch’s t -test) c Lung volume measurement of Yap1 , Wwtr1 iPCKO and littermate control lungs at the indicated stages. Data represents mean ± s.e.m. ( n = 5 for P7, n = 7 for P12, n = 7 for P18 controls, n = 5 for P18 mutant mice, two-tailed unpaired t -test). d Representative flow cytometry plots of EdU incorporation in EpCAM + cells from P7 Yap1 , Wwtr1 iPCKO and littermate control mice. e Diagrams showing flow cytometry cell cycle analysis of <t>CD31+</t> or EpCAM+ cells in P7 Yap1 , Wwtr1 iPCKO and littermate control lungs. Data represents mean ± s.e.m. ( n = 8 mice, NS: not significant, Unpaired two tailed student t -test or Welch’s t -test). f Three-dimensional reconstruction confocal images of P7 Yap1 , Wwtr1 iPCKO and littermate control lungs stained for SFTPC (green), EdU (red), and DAPI (blue). Quantitation of EdU positive and SFTPC positive cells is shown on the right. Data represents mean ± s.e.m. ( n = 4; unpaired two tailed student t -test). Scale bar, 30 µm
    Cd31 Monoclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cd31 monoclonal antibody/product/Thermo Fisher
    Average 95 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    cd31 monoclonal antibody - by Bioz Stars, 2020-09
    95/100 stars
      Buy from Supplier

    99
    Thermo Fisher anti mouse cd31 antibody
    The effect of AGS8 knockdown in cells sprouting from explant cultures of murine RPE-choroid. ( A ) AGS8 expression was determined by immunofluorescence staining of sprouting cells from explants. Sprouting cells on a coverslip were fixed with 4% PFA and stained with an anti-AGS8 antibody (red) and fluorescein labeled isolectin B4 (Vector Laboratories, Burlingame, CA))(green). Images were obtained with a fluorescence microscope. Representative images are shown from 10 specimens in 2 independent experiments with similar results. Scale bars are 100 μm. ( B ) The ratio of <t>CD31-positive</t> cells sprouting from choroid explant culture was assessed. Mouse choroid was isolated and cut into pieces and cultured on Matrigel-coated cover glass. Sprouting cells were collected on day 5 and stained with CD31-FITC antibody before processing for flow cytometric analysis. Negative control indicates cells stained with control IgG-FITC. ( C ) For AGS8 knockdown, AGS8 siRNA#1 was transfected to explanted cultures on days 2 and 3, and AGS8 mRNA expression on day 4 was determined by real-time PCR. Data are means ± s.e.m from four time-independent quaternary experiments. ** P
    Anti Mouse Cd31 Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti mouse cd31 antibody/product/Thermo Fisher
    Average 99 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    anti mouse cd31 antibody - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    Altered alveologenesis in PC-specific Yap1 and Wwtr1 mutant mice. a Three-dimensional reconstruction confocal images of Yap1 , Wwtr1 iPCKO and littermate control lungs stained for AQP5 (green), PDGFRβ (red), and PECAM1 (blue) at the indicated stages. Scale bar, 50 µm. b Quantitation of airspace volume in Yap1 , Wwtr1 iPCKO and control lung sections with three-dimensional reconstruction surface images. Data represents mean ± s.e.m. ( n = 4 mice; unpaired two tailed student t -test or Welch’s t -test) c Lung volume measurement of Yap1 , Wwtr1 iPCKO and littermate control lungs at the indicated stages. Data represents mean ± s.e.m. ( n = 5 for P7, n = 7 for P12, n = 7 for P18 controls, n = 5 for P18 mutant mice, two-tailed unpaired t -test). d Representative flow cytometry plots of EdU incorporation in EpCAM + cells from P7 Yap1 , Wwtr1 iPCKO and littermate control mice. e Diagrams showing flow cytometry cell cycle analysis of CD31+ or EpCAM+ cells in P7 Yap1 , Wwtr1 iPCKO and littermate control lungs. Data represents mean ± s.e.m. ( n = 8 mice, NS: not significant, Unpaired two tailed student t -test or Welch’s t -test). f Three-dimensional reconstruction confocal images of P7 Yap1 , Wwtr1 iPCKO and littermate control lungs stained for SFTPC (green), EdU (red), and DAPI (blue). Quantitation of EdU positive and SFTPC positive cells is shown on the right. Data represents mean ± s.e.m. ( n = 4; unpaired two tailed student t -test). Scale bar, 30 µm

    Journal: Nature Communications

    Article Title: Pulmonary pericytes regulate lung morphogenesis

    doi: 10.1038/s41467-018-04913-2

    Figure Lengend Snippet: Altered alveologenesis in PC-specific Yap1 and Wwtr1 mutant mice. a Three-dimensional reconstruction confocal images of Yap1 , Wwtr1 iPCKO and littermate control lungs stained for AQP5 (green), PDGFRβ (red), and PECAM1 (blue) at the indicated stages. Scale bar, 50 µm. b Quantitation of airspace volume in Yap1 , Wwtr1 iPCKO and control lung sections with three-dimensional reconstruction surface images. Data represents mean ± s.e.m. ( n = 4 mice; unpaired two tailed student t -test or Welch’s t -test) c Lung volume measurement of Yap1 , Wwtr1 iPCKO and littermate control lungs at the indicated stages. Data represents mean ± s.e.m. ( n = 5 for P7, n = 7 for P12, n = 7 for P18 controls, n = 5 for P18 mutant mice, two-tailed unpaired t -test). d Representative flow cytometry plots of EdU incorporation in EpCAM + cells from P7 Yap1 , Wwtr1 iPCKO and littermate control mice. e Diagrams showing flow cytometry cell cycle analysis of CD31+ or EpCAM+ cells in P7 Yap1 , Wwtr1 iPCKO and littermate control lungs. Data represents mean ± s.e.m. ( n = 8 mice, NS: not significant, Unpaired two tailed student t -test or Welch’s t -test). f Three-dimensional reconstruction confocal images of P7 Yap1 , Wwtr1 iPCKO and littermate control lungs stained for SFTPC (green), EdU (red), and DAPI (blue). Quantitation of EdU positive and SFTPC positive cells is shown on the right. Data represents mean ± s.e.m. ( n = 4; unpaired two tailed student t -test). Scale bar, 30 µm

    Article Snippet: For cell sorting from Yap1 ,Wwtr1 iPCKO mice and Angpt1 iPCKO mice, cells were immunostained with rat anti-TER-119-Pacific Blue (116232, BioLegend, 1:100), rat anti-CD45-Pacific Blue (103126, BioLegend, 1:100), rat anti-CD31-FITC (RM5201, Thermo Fisher, 1:50), rat anti-CD140a-PE (12–1401–81, eBioscience, 1:100), rat anti-CD326-PE/Cy7 (1:50), rat anti-CD140b-APC (17–1402–82, eBioscience, 1:25) for 30 min on ice.

    Techniques: Mutagenesis, Mouse Assay, Staining, Quantitation Assay, Two Tailed Test, Flow Cytometry, Cytometry, Cell Cycle Assay

    Characterisation of pulmonary pericytes during alveologenesis. a , b Three-dimensional reconstruction ( a ) and high magnification thin optical section ( b ) of confocal images with Airyscan detection showing AQP5-stained type 1 alveolar epithelial cells (green), PDGFRβ-stained pulmonary pericytes (PCs) (red) and PECAM1-stained endothelial cells (ECs) (blue) in lung at 4 weeks. Panels on the right show higher magnification of corresponding insets ( a ) or single channels ( b ). Scale bar, 30 µm ( a , left), 15 µm ( a , right), 10 µm ( b , left) and 5 µm ( b , right panels). c , d Three-dimensional reconstruction ( c ) and thin optical section ( d ) of confocal images showing SFTPC-stained, cuboidal AT2 cells (green), PDGFRβ-stained PCs (red) and RAGE-stained AT1 cells (blue) in lung at 4 weeks. Panels on the right show higher magnification of corresponding insets. Scale bar, 20 µm (left), 10 µm (right). e Quantitation of EC to PC ratio as measured in peripheral lung sections at different developmental stages. Data represents mean ± s.e.m. ( n = 5 for P7, n = 6 for P21 and Adult mice). f Scheme showing the time points of tamoxifen administration (P1–3) and analysis for the Pdgfrb(BAC)-CreERT2 R26-mT/mG mice. g – k High magnification images of Pdgfrb(BAC)-CreERT2 R26-mT/mG lung sections at indicated stages showing pulmonary GFP+ PCs (green), PECAM1+ ECs, PDGFRβ+ PCs, PDGFRα+ fibroblasts or αSMA+ bronchial smooth muscle cells/myofibroblasts (red, as indicated). Arrows in g indicate PC cell bodies, in g – k GFP-positive PDGFRβ+ cells ( g , h ), GFP-negative PDGFRα+ cells ( i ) or αSMA+ cells ( j , k ) are marked. Scale bar, 20 µm ( g , h ), 15 µm ( i , k ) and 30 µm ( j ). l RT-qPCR analysis of Pdgfrb (pericytes), Pecam1 (endothelium), and Sftpc (epithelium) expression in freshly sorted GFP+, CD31+ or EpCAM+ cells from P7 Pdgfrb(BAC)-CreERT2 R26-mT/mG lung. Data represents mean ± s.e.m. ( n = 4 mice)

    Journal: Nature Communications

    Article Title: Pulmonary pericytes regulate lung morphogenesis

    doi: 10.1038/s41467-018-04913-2

    Figure Lengend Snippet: Characterisation of pulmonary pericytes during alveologenesis. a , b Three-dimensional reconstruction ( a ) and high magnification thin optical section ( b ) of confocal images with Airyscan detection showing AQP5-stained type 1 alveolar epithelial cells (green), PDGFRβ-stained pulmonary pericytes (PCs) (red) and PECAM1-stained endothelial cells (ECs) (blue) in lung at 4 weeks. Panels on the right show higher magnification of corresponding insets ( a ) or single channels ( b ). Scale bar, 30 µm ( a , left), 15 µm ( a , right), 10 µm ( b , left) and 5 µm ( b , right panels). c , d Three-dimensional reconstruction ( c ) and thin optical section ( d ) of confocal images showing SFTPC-stained, cuboidal AT2 cells (green), PDGFRβ-stained PCs (red) and RAGE-stained AT1 cells (blue) in lung at 4 weeks. Panels on the right show higher magnification of corresponding insets. Scale bar, 20 µm (left), 10 µm (right). e Quantitation of EC to PC ratio as measured in peripheral lung sections at different developmental stages. Data represents mean ± s.e.m. ( n = 5 for P7, n = 6 for P21 and Adult mice). f Scheme showing the time points of tamoxifen administration (P1–3) and analysis for the Pdgfrb(BAC)-CreERT2 R26-mT/mG mice. g – k High magnification images of Pdgfrb(BAC)-CreERT2 R26-mT/mG lung sections at indicated stages showing pulmonary GFP+ PCs (green), PECAM1+ ECs, PDGFRβ+ PCs, PDGFRα+ fibroblasts or αSMA+ bronchial smooth muscle cells/myofibroblasts (red, as indicated). Arrows in g indicate PC cell bodies, in g – k GFP-positive PDGFRβ+ cells ( g , h ), GFP-negative PDGFRα+ cells ( i ) or αSMA+ cells ( j , k ) are marked. Scale bar, 20 µm ( g , h ), 15 µm ( i , k ) and 30 µm ( j ). l RT-qPCR analysis of Pdgfrb (pericytes), Pecam1 (endothelium), and Sftpc (epithelium) expression in freshly sorted GFP+, CD31+ or EpCAM+ cells from P7 Pdgfrb(BAC)-CreERT2 R26-mT/mG lung. Data represents mean ± s.e.m. ( n = 4 mice)

    Article Snippet: For cell sorting from Yap1 ,Wwtr1 iPCKO mice and Angpt1 iPCKO mice, cells were immunostained with rat anti-TER-119-Pacific Blue (116232, BioLegend, 1:100), rat anti-CD45-Pacific Blue (103126, BioLegend, 1:100), rat anti-CD31-FITC (RM5201, Thermo Fisher, 1:50), rat anti-CD140a-PE (12–1401–81, eBioscience, 1:100), rat anti-CD326-PE/Cy7 (1:50), rat anti-CD140b-APC (17–1402–82, eBioscience, 1:25) for 30 min on ice.

    Techniques: Staining, Quantitation Assay, Mouse Assay, Quantitative RT-PCR, Expressing

    Pericyte-derived Angpt1 controls alveologenesis. a RT-qPCR analysis of Angpt1 and Tie2 / Tek expression in freshly sorted lung GFP+, CD31+ or EpCAM+ cells from P7 Pdgfrb(BAC)-CreERT2 R26-mT/mG mice. Data represents mean ± s.e.m. ( n = 4 mice). b High magnification images of P10 Angpt1 GFP lungs stained for GFP (green), PDGFRβ (red), and PDGFRα (blue). Arrows indicate GFP and PDGFRβ double positive pericytes. Scale bar, 15 µm. c RT-qPCR analysis of Angpt1 expression in freshly sorted PDGFRβ+ cells from P7 Yap1 , Wwtr1 iPCKO and control lungs. Data represents mean ± s.e.m. ( n = 4 mice, two-tailed unpaired t -test). d Angpt1 expression in cultured Verteporfin (VP)-treated (48 h) and control pericytes. Data represents mean ± s.e.m. ( n = 4, Welch’s t -test). e Expression of the indicated transcripts in freshly sorted CD31+ cells from P7 Yap1 , Wwtr1 iPCKO and control lungs. Data represents mean ± s.e.m. ( n = 4 mice, NS not significant, two-tailed unpaired t -test). f – h Western blot analysis of Angpt1 protein ( f ; n = 2 controls and 4 mutant mice) and of total and phospho-Tie2 (pTie2) in P12 Yap1 , Wwtr1 iPCKO and control total lung lysates ( g , n = 3 controls and 5 mutants). Molecular weight marker (kDa) is indicated. Relative quantification of signals is shown in h . Two-tailed unpaired t -test. i Scheme showing the time points of tamoxifen administration and analysis for Angpt1 iPCKO mice. j , k 3D reconstruction confocal images of P12 Angpt1 iPCKO and littermate control lungs stained for AQP5 (green), PDGFRβ (red), and PECAM1 (blue). Panels in k show higher magnification of PECAM1 staining. Scale bar, 50 µm ( j ) and 30 µm ( k ). l Quantitation of airspace volume in P12 Angpt1 iPCKO and littermate control lung sections with 3D reconstruction surface images. Data represents mean ± s.e.m. ( n = 4 mice; p

    Journal: Nature Communications

    Article Title: Pulmonary pericytes regulate lung morphogenesis

    doi: 10.1038/s41467-018-04913-2

    Figure Lengend Snippet: Pericyte-derived Angpt1 controls alveologenesis. a RT-qPCR analysis of Angpt1 and Tie2 / Tek expression in freshly sorted lung GFP+, CD31+ or EpCAM+ cells from P7 Pdgfrb(BAC)-CreERT2 R26-mT/mG mice. Data represents mean ± s.e.m. ( n = 4 mice). b High magnification images of P10 Angpt1 GFP lungs stained for GFP (green), PDGFRβ (red), and PDGFRα (blue). Arrows indicate GFP and PDGFRβ double positive pericytes. Scale bar, 15 µm. c RT-qPCR analysis of Angpt1 expression in freshly sorted PDGFRβ+ cells from P7 Yap1 , Wwtr1 iPCKO and control lungs. Data represents mean ± s.e.m. ( n = 4 mice, two-tailed unpaired t -test). d Angpt1 expression in cultured Verteporfin (VP)-treated (48 h) and control pericytes. Data represents mean ± s.e.m. ( n = 4, Welch’s t -test). e Expression of the indicated transcripts in freshly sorted CD31+ cells from P7 Yap1 , Wwtr1 iPCKO and control lungs. Data represents mean ± s.e.m. ( n = 4 mice, NS not significant, two-tailed unpaired t -test). f – h Western blot analysis of Angpt1 protein ( f ; n = 2 controls and 4 mutant mice) and of total and phospho-Tie2 (pTie2) in P12 Yap1 , Wwtr1 iPCKO and control total lung lysates ( g , n = 3 controls and 5 mutants). Molecular weight marker (kDa) is indicated. Relative quantification of signals is shown in h . Two-tailed unpaired t -test. i Scheme showing the time points of tamoxifen administration and analysis for Angpt1 iPCKO mice. j , k 3D reconstruction confocal images of P12 Angpt1 iPCKO and littermate control lungs stained for AQP5 (green), PDGFRβ (red), and PECAM1 (blue). Panels in k show higher magnification of PECAM1 staining. Scale bar, 50 µm ( j ) and 30 µm ( k ). l Quantitation of airspace volume in P12 Angpt1 iPCKO and littermate control lung sections with 3D reconstruction surface images. Data represents mean ± s.e.m. ( n = 4 mice; p

    Article Snippet: For cell sorting from Yap1 ,Wwtr1 iPCKO mice and Angpt1 iPCKO mice, cells were immunostained with rat anti-TER-119-Pacific Blue (116232, BioLegend, 1:100), rat anti-CD45-Pacific Blue (103126, BioLegend, 1:100), rat anti-CD31-FITC (RM5201, Thermo Fisher, 1:50), rat anti-CD140a-PE (12–1401–81, eBioscience, 1:100), rat anti-CD326-PE/Cy7 (1:50), rat anti-CD140b-APC (17–1402–82, eBioscience, 1:25) for 30 min on ice.

    Techniques: Derivative Assay, Quantitative RT-PCR, Expressing, Mouse Assay, Staining, Two Tailed Test, Cell Culture, Western Blot, Mutagenesis, Molecular Weight, Marker, Quantitation Assay

    Altered alveologenesis in PC-specific Yap1 and Wwtr1 mutant mice. a Three-dimensional reconstruction confocal images of Yap1 , Wwtr1 iPCKO and littermate control lungs stained for AQP5 (green), PDGFRβ (red), and PECAM1 (blue) at the indicated stages. Scale bar, 50 µm. b Quantitation of airspace volume in Yap1 , Wwtr1 iPCKO and control lung sections with three-dimensional reconstruction surface images. Data represents mean ± s.e.m. ( n = 4 mice; unpaired two tailed student t -test or Welch’s t -test) c Lung volume measurement of Yap1 , Wwtr1 iPCKO and littermate control lungs at the indicated stages. Data represents mean ± s.e.m. ( n = 5 for P7, n = 7 for P12, n = 7 for P18 controls, n = 5 for P18 mutant mice, two-tailed unpaired t -test). d Representative flow cytometry plots of EdU incorporation in EpCAM + cells from P7 Yap1 , Wwtr1 iPCKO and littermate control mice. e Diagrams showing flow cytometry cell cycle analysis of CD31+ or EpCAM+ cells in P7 Yap1 , Wwtr1 iPCKO and littermate control lungs. Data represents mean ± s.e.m. ( n = 8 mice, NS: not significant, Unpaired two tailed student t -test or Welch’s t -test). f Three-dimensional reconstruction confocal images of P7 Yap1 , Wwtr1 iPCKO and littermate control lungs stained for SFTPC (green), EdU (red), and DAPI (blue). Quantitation of EdU positive and SFTPC positive cells is shown on the right. Data represents mean ± s.e.m. ( n = 4; unpaired two tailed student t -test). Scale bar, 30 µm

    Journal: Nature Communications

    Article Title: Pulmonary pericytes regulate lung morphogenesis

    doi: 10.1038/s41467-018-04913-2

    Figure Lengend Snippet: Altered alveologenesis in PC-specific Yap1 and Wwtr1 mutant mice. a Three-dimensional reconstruction confocal images of Yap1 , Wwtr1 iPCKO and littermate control lungs stained for AQP5 (green), PDGFRβ (red), and PECAM1 (blue) at the indicated stages. Scale bar, 50 µm. b Quantitation of airspace volume in Yap1 , Wwtr1 iPCKO and control lung sections with three-dimensional reconstruction surface images. Data represents mean ± s.e.m. ( n = 4 mice; unpaired two tailed student t -test or Welch’s t -test) c Lung volume measurement of Yap1 , Wwtr1 iPCKO and littermate control lungs at the indicated stages. Data represents mean ± s.e.m. ( n = 5 for P7, n = 7 for P12, n = 7 for P18 controls, n = 5 for P18 mutant mice, two-tailed unpaired t -test). d Representative flow cytometry plots of EdU incorporation in EpCAM + cells from P7 Yap1 , Wwtr1 iPCKO and littermate control mice. e Diagrams showing flow cytometry cell cycle analysis of CD31+ or EpCAM+ cells in P7 Yap1 , Wwtr1 iPCKO and littermate control lungs. Data represents mean ± s.e.m. ( n = 8 mice, NS: not significant, Unpaired two tailed student t -test or Welch’s t -test). f Three-dimensional reconstruction confocal images of P7 Yap1 , Wwtr1 iPCKO and littermate control lungs stained for SFTPC (green), EdU (red), and DAPI (blue). Quantitation of EdU positive and SFTPC positive cells is shown on the right. Data represents mean ± s.e.m. ( n = 4; unpaired two tailed student t -test). Scale bar, 30 µm

    Article Snippet: For cell sorting from Yap1 , Wwtr1 iPCKO mice and Angpt1 iPCKO mice, cells were immunostained with rat anti-TER-119-Pacific Blue (116232, BioLegend, 1:100), rat anti-CD45-Pacific Blue (103126, BioLegend, 1:100), rat anti-CD31-FITC (RM5201, Thermo Fisher, 1:50), rat anti-CD140a-PE (12–1401–81, eBioscience, 1:100), rat anti-CD326-PE/Cy7 (1:50), rat anti-CD140b-APC (17–1402–82, eBioscience, 1:25) for 30 min on ice.

    Techniques: Mutagenesis, Mouse Assay, Staining, Quantitation Assay, Two Tailed Test, Flow Cytometry, Cytometry, Cell Cycle Assay

    Characterisation of pulmonary pericytes during alveologenesis. a , b Three-dimensional reconstruction ( a ) and high magnification thin optical section ( b ) of confocal images with Airyscan detection showing AQP5-stained type 1 alveolar epithelial cells (green), PDGFRβ-stained pulmonary pericytes (PCs) (red) and PECAM1-stained endothelial cells (ECs) (blue) in lung at 4 weeks. Panels on the right show higher magnification of corresponding insets ( a ) or single channels ( b ). Scale bar, 30 µm ( a , left), 15 µm ( a , right), 10 µm ( b , left) and 5 µm ( b , right panels). c , d Three-dimensional reconstruction ( c ) and thin optical section ( d ) of confocal images showing SFTPC-stained, cuboidal AT2 cells (green), PDGFRβ-stained PCs (red) and RAGE-stained AT1 cells (blue) in lung at 4 weeks. Panels on the right show higher magnification of corresponding insets. Scale bar, 20 µm (left), 10 µm (right). e Quantitation of EC to PC ratio as measured in peripheral lung sections at different developmental stages. Data represents mean ± s.e.m. ( n = 5 for P7, n = 6 for P21 and Adult mice). f Scheme showing the time points of tamoxifen administration (P1–3) and analysis for the Pdgfrb(BAC)-CreERT2 R26-mT/mG mice. g – k High magnification images of Pdgfrb(BAC)-CreERT2 R26-mT/mG lung sections at indicated stages showing pulmonary GFP+ PCs (green), PECAM1+ ECs, PDGFRβ+ PCs, PDGFRα+ fibroblasts or αSMA+ bronchial smooth muscle cells/myofibroblasts (red, as indicated). Arrows in g indicate PC cell bodies, in g – k GFP-positive PDGFRβ+ cells ( g , h ), GFP-negative PDGFRα+ cells ( i ) or αSMA+ cells ( j , k ) are marked. Scale bar, 20 µm ( g , h ), 15 µm ( i , k ) and 30 µm ( j ). l RT-qPCR analysis of Pdgfrb (pericytes), Pecam1 (endothelium), and Sftpc (epithelium) expression in freshly sorted GFP+, CD31+ or EpCAM+ cells from P7 Pdgfrb(BAC)-CreERT2 R26-mT/mG lung. Data represents mean ± s.e.m. ( n = 4 mice)

    Journal: Nature Communications

    Article Title: Pulmonary pericytes regulate lung morphogenesis

    doi: 10.1038/s41467-018-04913-2

    Figure Lengend Snippet: Characterisation of pulmonary pericytes during alveologenesis. a , b Three-dimensional reconstruction ( a ) and high magnification thin optical section ( b ) of confocal images with Airyscan detection showing AQP5-stained type 1 alveolar epithelial cells (green), PDGFRβ-stained pulmonary pericytes (PCs) (red) and PECAM1-stained endothelial cells (ECs) (blue) in lung at 4 weeks. Panels on the right show higher magnification of corresponding insets ( a ) or single channels ( b ). Scale bar, 30 µm ( a , left), 15 µm ( a , right), 10 µm ( b , left) and 5 µm ( b , right panels). c , d Three-dimensional reconstruction ( c ) and thin optical section ( d ) of confocal images showing SFTPC-stained, cuboidal AT2 cells (green), PDGFRβ-stained PCs (red) and RAGE-stained AT1 cells (blue) in lung at 4 weeks. Panels on the right show higher magnification of corresponding insets. Scale bar, 20 µm (left), 10 µm (right). e Quantitation of EC to PC ratio as measured in peripheral lung sections at different developmental stages. Data represents mean ± s.e.m. ( n = 5 for P7, n = 6 for P21 and Adult mice). f Scheme showing the time points of tamoxifen administration (P1–3) and analysis for the Pdgfrb(BAC)-CreERT2 R26-mT/mG mice. g – k High magnification images of Pdgfrb(BAC)-CreERT2 R26-mT/mG lung sections at indicated stages showing pulmonary GFP+ PCs (green), PECAM1+ ECs, PDGFRβ+ PCs, PDGFRα+ fibroblasts or αSMA+ bronchial smooth muscle cells/myofibroblasts (red, as indicated). Arrows in g indicate PC cell bodies, in g – k GFP-positive PDGFRβ+ cells ( g , h ), GFP-negative PDGFRα+ cells ( i ) or αSMA+ cells ( j , k ) are marked. Scale bar, 20 µm ( g , h ), 15 µm ( i , k ) and 30 µm ( j ). l RT-qPCR analysis of Pdgfrb (pericytes), Pecam1 (endothelium), and Sftpc (epithelium) expression in freshly sorted GFP+, CD31+ or EpCAM+ cells from P7 Pdgfrb(BAC)-CreERT2 R26-mT/mG lung. Data represents mean ± s.e.m. ( n = 4 mice)

    Article Snippet: For cell sorting from Yap1 , Wwtr1 iPCKO mice and Angpt1 iPCKO mice, cells were immunostained with rat anti-TER-119-Pacific Blue (116232, BioLegend, 1:100), rat anti-CD45-Pacific Blue (103126, BioLegend, 1:100), rat anti-CD31-FITC (RM5201, Thermo Fisher, 1:50), rat anti-CD140a-PE (12–1401–81, eBioscience, 1:100), rat anti-CD326-PE/Cy7 (1:50), rat anti-CD140b-APC (17–1402–82, eBioscience, 1:25) for 30 min on ice.

    Techniques: Staining, Quantitation Assay, Mouse Assay, Quantitative RT-PCR, Expressing

    Pericyte-derived Angpt1 controls alveologenesis. a RT-qPCR analysis of Angpt1 and Tie2 / Tek expression in freshly sorted lung GFP+, CD31+ or EpCAM+ cells from P7 Pdgfrb(BAC)-CreERT2 R26-mT/mG mice. Data represents mean ± s.e.m. ( n = 4 mice). b High magnification images of P10 Angpt1 GFP lungs stained for GFP (green), PDGFRβ (red), and PDGFRα (blue). Arrows indicate GFP and PDGFRβ double positive pericytes. Scale bar, 15 µm. c RT-qPCR analysis of Angpt1 expression in freshly sorted PDGFRβ+ cells from P7 Yap1 , Wwtr1 iPCKO and control lungs. Data represents mean ± s.e.m. ( n = 4 mice, two-tailed unpaired t -test). d Angpt1 expression in cultured Verteporfin (VP)-treated (48 h) and control pericytes. Data represents mean ± s.e.m. ( n = 4, Welch’s t -test). e Expression of the indicated transcripts in freshly sorted CD31+ cells from P7 Yap1 , Wwtr1 iPCKO and control lungs. Data represents mean ± s.e.m. ( n = 4 mice, NS not significant, two-tailed unpaired t -test). f – h Western blot analysis of Angpt1 protein ( f ; n = 2 controls and 4 mutant mice) and of total and phospho-Tie2 (pTie2) in P12 Yap1 , Wwtr1 iPCKO and control total lung lysates ( g , n = 3 controls and 5 mutants). Molecular weight marker (kDa) is indicated. Relative quantification of signals is shown in h . Two-tailed unpaired t -test. i Scheme showing the time points of tamoxifen administration and analysis for Angpt1 iPCKO mice. j , k 3D reconstruction confocal images of P12 Angpt1 iPCKO and littermate control lungs stained for AQP5 (green), PDGFRβ (red), and PECAM1 (blue). Panels in k show higher magnification of PECAM1 staining. Scale bar, 50 µm ( j ) and 30 µm ( k ). l Quantitation of airspace volume in P12 Angpt1 iPCKO and littermate control lung sections with 3D reconstruction surface images. Data represents mean ± s.e.m. ( n = 4 mice; p

    Journal: Nature Communications

    Article Title: Pulmonary pericytes regulate lung morphogenesis

    doi: 10.1038/s41467-018-04913-2

    Figure Lengend Snippet: Pericyte-derived Angpt1 controls alveologenesis. a RT-qPCR analysis of Angpt1 and Tie2 / Tek expression in freshly sorted lung GFP+, CD31+ or EpCAM+ cells from P7 Pdgfrb(BAC)-CreERT2 R26-mT/mG mice. Data represents mean ± s.e.m. ( n = 4 mice). b High magnification images of P10 Angpt1 GFP lungs stained for GFP (green), PDGFRβ (red), and PDGFRα (blue). Arrows indicate GFP and PDGFRβ double positive pericytes. Scale bar, 15 µm. c RT-qPCR analysis of Angpt1 expression in freshly sorted PDGFRβ+ cells from P7 Yap1 , Wwtr1 iPCKO and control lungs. Data represents mean ± s.e.m. ( n = 4 mice, two-tailed unpaired t -test). d Angpt1 expression in cultured Verteporfin (VP)-treated (48 h) and control pericytes. Data represents mean ± s.e.m. ( n = 4, Welch’s t -test). e Expression of the indicated transcripts in freshly sorted CD31+ cells from P7 Yap1 , Wwtr1 iPCKO and control lungs. Data represents mean ± s.e.m. ( n = 4 mice, NS not significant, two-tailed unpaired t -test). f – h Western blot analysis of Angpt1 protein ( f ; n = 2 controls and 4 mutant mice) and of total and phospho-Tie2 (pTie2) in P12 Yap1 , Wwtr1 iPCKO and control total lung lysates ( g , n = 3 controls and 5 mutants). Molecular weight marker (kDa) is indicated. Relative quantification of signals is shown in h . Two-tailed unpaired t -test. i Scheme showing the time points of tamoxifen administration and analysis for Angpt1 iPCKO mice. j , k 3D reconstruction confocal images of P12 Angpt1 iPCKO and littermate control lungs stained for AQP5 (green), PDGFRβ (red), and PECAM1 (blue). Panels in k show higher magnification of PECAM1 staining. Scale bar, 50 µm ( j ) and 30 µm ( k ). l Quantitation of airspace volume in P12 Angpt1 iPCKO and littermate control lung sections with 3D reconstruction surface images. Data represents mean ± s.e.m. ( n = 4 mice; p

    Article Snippet: For cell sorting from Yap1 , Wwtr1 iPCKO mice and Angpt1 iPCKO mice, cells were immunostained with rat anti-TER-119-Pacific Blue (116232, BioLegend, 1:100), rat anti-CD45-Pacific Blue (103126, BioLegend, 1:100), rat anti-CD31-FITC (RM5201, Thermo Fisher, 1:50), rat anti-CD140a-PE (12–1401–81, eBioscience, 1:100), rat anti-CD326-PE/Cy7 (1:50), rat anti-CD140b-APC (17–1402–82, eBioscience, 1:25) for 30 min on ice.

    Techniques: Derivative Assay, Quantitative RT-PCR, Expressing, Mouse Assay, Staining, Two Tailed Test, Cell Culture, Western Blot, Mutagenesis, Molecular Weight, Marker, Quantitation Assay

    The effect of AGS8 knockdown in cells sprouting from explant cultures of murine RPE-choroid. ( A ) AGS8 expression was determined by immunofluorescence staining of sprouting cells from explants. Sprouting cells on a coverslip were fixed with 4% PFA and stained with an anti-AGS8 antibody (red) and fluorescein labeled isolectin B4 (Vector Laboratories, Burlingame, CA))(green). Images were obtained with a fluorescence microscope. Representative images are shown from 10 specimens in 2 independent experiments with similar results. Scale bars are 100 μm. ( B ) The ratio of CD31-positive cells sprouting from choroid explant culture was assessed. Mouse choroid was isolated and cut into pieces and cultured on Matrigel-coated cover glass. Sprouting cells were collected on day 5 and stained with CD31-FITC antibody before processing for flow cytometric analysis. Negative control indicates cells stained with control IgG-FITC. ( C ) For AGS8 knockdown, AGS8 siRNA#1 was transfected to explanted cultures on days 2 and 3, and AGS8 mRNA expression on day 4 was determined by real-time PCR. Data are means ± s.e.m from four time-independent quaternary experiments. ** P

    Journal: Scientific Reports

    Article Title: Activator of G-protein signaling 8 is involved in VEGF-induced choroidal neovascularization

    doi: 10.1038/s41598-018-38067-4

    Figure Lengend Snippet: The effect of AGS8 knockdown in cells sprouting from explant cultures of murine RPE-choroid. ( A ) AGS8 expression was determined by immunofluorescence staining of sprouting cells from explants. Sprouting cells on a coverslip were fixed with 4% PFA and stained with an anti-AGS8 antibody (red) and fluorescein labeled isolectin B4 (Vector Laboratories, Burlingame, CA))(green). Images were obtained with a fluorescence microscope. Representative images are shown from 10 specimens in 2 independent experiments with similar results. Scale bars are 100 μm. ( B ) The ratio of CD31-positive cells sprouting from choroid explant culture was assessed. Mouse choroid was isolated and cut into pieces and cultured on Matrigel-coated cover glass. Sprouting cells were collected on day 5 and stained with CD31-FITC antibody before processing for flow cytometric analysis. Negative control indicates cells stained with control IgG-FITC. ( C ) For AGS8 knockdown, AGS8 siRNA#1 was transfected to explanted cultures on days 2 and 3, and AGS8 mRNA expression on day 4 was determined by real-time PCR. Data are means ± s.e.m from four time-independent quaternary experiments. ** P

    Article Snippet: On the day of experiments, methanol was replaced by PBS and 0.5% Triton X (PBST) at room temperature, and PBST with 5% goat serum (Jackson ImmunoResearch, West Grove, PA) blocking buffer for 30 min, before incubation with anti-mouse CD31 antibody (rat, 1:500; Thermo Fisher), or anti-mouse/human AGS8 antibody (rabbit, 1:200) overnight at 4 °C.

    Techniques: Expressing, Immunofluorescence, Staining, Labeling, Plasmid Preparation, Fluorescence, Microscopy, Isolation, Cell Culture, Flow Cytometry, Negative Control, Transfection, Real-time Polymerase Chain Reaction

    Upregulation of AGS8 mRNA expression in choroidal EC in a laser-induced CNV experiment. ( A ) Picture of the flat-mounted choroid 2 days post-laser-induced CNV. Mouse retinas in each eye were subjected to laser photocoagulation for induction of experimental CNV with an argon laser photocoagulator. Dissected mouse RPE-choroid was immunostained with CD31 (red; Cy3) and AGS8 (green; Alexa488) antibodies. The arrow indicates CD31-positive cells in the laser-induced CNV area, whereas the arrowhead indicates CD31-positive cells outside of the laser-induced lesions. ( B ) AGS8 mRNA expression in CNV area. Two days after laser-induced CNV, mouse eyes were dissected, RPE-choroid was isolated, the lesion area was dissected, and total RNA was purified. Real-time PCR was performed to evaluate laser-induced AGS8 mRNA expression. Data are means ± s.e.m from four time-independent experiments. * P

    Journal: Scientific Reports

    Article Title: Activator of G-protein signaling 8 is involved in VEGF-induced choroidal neovascularization

    doi: 10.1038/s41598-018-38067-4

    Figure Lengend Snippet: Upregulation of AGS8 mRNA expression in choroidal EC in a laser-induced CNV experiment. ( A ) Picture of the flat-mounted choroid 2 days post-laser-induced CNV. Mouse retinas in each eye were subjected to laser photocoagulation for induction of experimental CNV with an argon laser photocoagulator. Dissected mouse RPE-choroid was immunostained with CD31 (red; Cy3) and AGS8 (green; Alexa488) antibodies. The arrow indicates CD31-positive cells in the laser-induced CNV area, whereas the arrowhead indicates CD31-positive cells outside of the laser-induced lesions. ( B ) AGS8 mRNA expression in CNV area. Two days after laser-induced CNV, mouse eyes were dissected, RPE-choroid was isolated, the lesion area was dissected, and total RNA was purified. Real-time PCR was performed to evaluate laser-induced AGS8 mRNA expression. Data are means ± s.e.m from four time-independent experiments. * P

    Article Snippet: On the day of experiments, methanol was replaced by PBS and 0.5% Triton X (PBST) at room temperature, and PBST with 5% goat serum (Jackson ImmunoResearch, West Grove, PA) blocking buffer for 30 min, before incubation with anti-mouse CD31 antibody (rat, 1:500; Thermo Fisher), or anti-mouse/human AGS8 antibody (rabbit, 1:200) overnight at 4 °C.

    Techniques: Expressing, Isolation, Purification, Real-time Polymerase Chain Reaction

    The effect of AGS8 knockdown in cells sprouting from explant cultures of murine RPE-choroid. ( A ) AGS8 expression was determined by immunofluorescence staining of sprouting cells from explants. Sprouting cells on a coverslip were fixed with 4% PFA and stained with an anti-AGS8 antibody (red) and fluorescein labeled isolectin B4 (Vector Laboratories, Burlingame, CA))(green). Images were obtained with a fluorescence microscope. Representative images are shown from 10 specimens in 2 independent experiments with similar results. Scale bars are 100 μm. ( B ) The ratio of CD31-positive cells sprouting from choroid explant culture was assessed. Mouse choroid was isolated and cut into pieces and cultured on Matrigel-coated cover glass. Sprouting cells were collected on day 5 and stained with CD31-FITC antibody before processing for flow cytometric analysis. Negative control indicates cells stained with control IgG-FITC. ( C ) For AGS8 knockdown, AGS8 siRNA#1 was transfected to explanted cultures on days 2 and 3, and AGS8 mRNA expression on day 4 was determined by real-time PCR. Data are means ± s.e.m from four time-independent quaternary experiments. ** P

    Journal: Scientific Reports

    Article Title: Activator of G-protein signaling 8 is involved in VEGF-induced choroidal neovascularization

    doi: 10.1038/s41598-018-38067-4

    Figure Lengend Snippet: The effect of AGS8 knockdown in cells sprouting from explant cultures of murine RPE-choroid. ( A ) AGS8 expression was determined by immunofluorescence staining of sprouting cells from explants. Sprouting cells on a coverslip were fixed with 4% PFA and stained with an anti-AGS8 antibody (red) and fluorescein labeled isolectin B4 (Vector Laboratories, Burlingame, CA))(green). Images were obtained with a fluorescence microscope. Representative images are shown from 10 specimens in 2 independent experiments with similar results. Scale bars are 100 μm. ( B ) The ratio of CD31-positive cells sprouting from choroid explant culture was assessed. Mouse choroid was isolated and cut into pieces and cultured on Matrigel-coated cover glass. Sprouting cells were collected on day 5 and stained with CD31-FITC antibody before processing for flow cytometric analysis. Negative control indicates cells stained with control IgG-FITC. ( C ) For AGS8 knockdown, AGS8 siRNA#1 was transfected to explanted cultures on days 2 and 3, and AGS8 mRNA expression on day 4 was determined by real-time PCR. Data are means ± s.e.m from four time-independent quaternary experiments. ** P

    Article Snippet: On the day of experiments, methanol was replaced by PBS and 0.5% Triton X (PBST) at room temperature, and PBST with 5% goat serum (Jackson ImmunoResearch, West Grove, PA) blocking buffer for 30 min, before incubation with anti-mouse CD31 antibody (rat, 1:500; Thermo Fisher), or anti-mouse/human AGS8 antibody (rabbit, 1:200) overnight at 4 °C.

    Techniques: Expressing, Immunofluorescence, Staining, Labeling, Plasmid Preparation, Fluorescence, Microscopy, Isolation, Cell Culture, Flow Cytometry, Negative Control, Transfection, Real-time Polymerase Chain Reaction

    Upregulation of AGS8 mRNA expression in choroidal EC in a laser-induced CNV experiment. ( A ) Picture of the flat-mounted choroid 2 days post-laser-induced CNV. Mouse retinas in each eye were subjected to laser photocoagulation for induction of experimental CNV with an argon laser photocoagulator. Dissected mouse RPE-choroid was immunostained with CD31 (red; Cy3) and AGS8 (green; Alexa488) antibodies. The arrow indicates CD31-positive cells in the laser-induced CNV area, whereas the arrowhead indicates CD31-positive cells outside of the laser-induced lesions. ( B ) AGS8 mRNA expression in CNV area. Two days after laser-induced CNV, mouse eyes were dissected, RPE-choroid was isolated, the lesion area was dissected, and total RNA was purified. Real-time PCR was performed to evaluate laser-induced AGS8 mRNA expression. Data are means ± s.e.m from four time-independent experiments. * P

    Journal: Scientific Reports

    Article Title: Activator of G-protein signaling 8 is involved in VEGF-induced choroidal neovascularization

    doi: 10.1038/s41598-018-38067-4

    Figure Lengend Snippet: Upregulation of AGS8 mRNA expression in choroidal EC in a laser-induced CNV experiment. ( A ) Picture of the flat-mounted choroid 2 days post-laser-induced CNV. Mouse retinas in each eye were subjected to laser photocoagulation for induction of experimental CNV with an argon laser photocoagulator. Dissected mouse RPE-choroid was immunostained with CD31 (red; Cy3) and AGS8 (green; Alexa488) antibodies. The arrow indicates CD31-positive cells in the laser-induced CNV area, whereas the arrowhead indicates CD31-positive cells outside of the laser-induced lesions. ( B ) AGS8 mRNA expression in CNV area. Two days after laser-induced CNV, mouse eyes were dissected, RPE-choroid was isolated, the lesion area was dissected, and total RNA was purified. Real-time PCR was performed to evaluate laser-induced AGS8 mRNA expression. Data are means ± s.e.m from four time-independent experiments. * P

    Article Snippet: On the day of experiments, methanol was replaced by PBS and 0.5% Triton X (PBST) at room temperature, and PBST with 5% goat serum (Jackson ImmunoResearch, West Grove, PA) blocking buffer for 30 min, before incubation with anti-mouse CD31 antibody (rat, 1:500; Thermo Fisher), or anti-mouse/human AGS8 antibody (rabbit, 1:200) overnight at 4 °C.

    Techniques: Expressing, Isolation, Purification, Real-time Polymerase Chain Reaction