anti igg1  (Abcam)

 
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    Anti IgG1 antibody
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    ab170461
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    Structured Review

    Abcam anti igg1
    Effects of TLR ‐7 agonist R848 in transgenic mice. A , Detection of GFP in bone marrow–derived macrophages ( BMM s) from mTLR ‐7−/− (knockout [ KO ]) mice and transgenic (Tg) mice by flow cytometric analysis, after staining with anti‐ CD 11b antibody. At least 2 independent experiments were performed. B , Interleukin‐6 ( IL ‐6) and IL ‐12p40 levels in BMM s from knockout mice and transgenic mice left unstimulated or stimulated with R848. After 24 hours of incubation, IL ‐6 and IL ‐12p40 were detected in culture medium by enzyme‐linked immunosorbent assay ( ELISA ). Results are from triplicate wells. At least 2 independent experiments were performed. C , Weight of SMG s, kidneys, lungs, and liver in 8‐week‐old knockout, wild‐type ( WT ), and transgenic mice treated with topical R848 for 4 weeks (n = 8 per group). D and F , H E‐stained ( D ) and Masson's trichrome–stained ( F ) sections of SMG s, pancreas, kidneys, lungs, and liver from representative 8‐week‐old R848‐treated knockout, WT , and transgenic mice. Bars = 100 μm. E and G , Focus score ( E ) and fibrosis score ( G ) for each organ in 8‐week‐old knockout, WT , and transgenic mice left untreated or treated with R848 (n = 8 per group). The fibrosis score was calculated from Masson's trichrome staining as described in Patients and Methods. HPF = high‐power field. H , Serial sections of SMG s from a representative 8‐week‐old R848‐treated transgenic mouse, stained with H E and for <t>IgG1,</t> TLR ‐7, CD 206, CD 317, and IL ‐33. Mayer's hematoxylin (blue) counterstained; bars = 100 μm. I , Serum IgG, IgG1, and IgG2a levels in knockout, WT , and transgenic mice (n = 10 per group) before and after R848 treatment, as determined by ELISA . Symbols represent individual mice; horizontal lines show the mean. J , Serum IL ‐33 levels, determined by ELISA , in knockout, WT , and transgenic mice left untreated or treated with R848 (n = 10 per group). In B , C , E , G , and J , bars show the mean ± SD . * = P

    https://www.bioz.com/result/anti igg1/product/Abcam
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    anti igg1 - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "Activated M2 Macrophages Contribute to the Pathogenesis of IgG4‐Related Disease via Toll‐like Receptor 7/Interleukin‐33 Signaling"

    Article Title: Activated M2 Macrophages Contribute to the Pathogenesis of IgG4‐Related Disease via Toll‐like Receptor 7/Interleukin‐33 Signaling

    Journal: Arthritis & Rheumatology (Hoboken, N.j.)

    doi: 10.1002/art.41052

    Effects of TLR ‐7 agonist R848 in transgenic mice. A , Detection of GFP in bone marrow–derived macrophages ( BMM s) from mTLR ‐7−/− (knockout [ KO ]) mice and transgenic (Tg) mice by flow cytometric analysis, after staining with anti‐ CD 11b antibody. At least 2 independent experiments were performed. B , Interleukin‐6 ( IL ‐6) and IL ‐12p40 levels in BMM s from knockout mice and transgenic mice left unstimulated or stimulated with R848. After 24 hours of incubation, IL ‐6 and IL ‐12p40 were detected in culture medium by enzyme‐linked immunosorbent assay ( ELISA ). Results are from triplicate wells. At least 2 independent experiments were performed. C , Weight of SMG s, kidneys, lungs, and liver in 8‐week‐old knockout, wild‐type ( WT ), and transgenic mice treated with topical R848 for 4 weeks (n = 8 per group). D and F , H E‐stained ( D ) and Masson's trichrome–stained ( F ) sections of SMG s, pancreas, kidneys, lungs, and liver from representative 8‐week‐old R848‐treated knockout, WT , and transgenic mice. Bars = 100 μm. E and G , Focus score ( E ) and fibrosis score ( G ) for each organ in 8‐week‐old knockout, WT , and transgenic mice left untreated or treated with R848 (n = 8 per group). The fibrosis score was calculated from Masson's trichrome staining as described in Patients and Methods. HPF = high‐power field. H , Serial sections of SMG s from a representative 8‐week‐old R848‐treated transgenic mouse, stained with H E and for IgG1, TLR ‐7, CD 206, CD 317, and IL ‐33. Mayer's hematoxylin (blue) counterstained; bars = 100 μm. I , Serum IgG, IgG1, and IgG2a levels in knockout, WT , and transgenic mice (n = 10 per group) before and after R848 treatment, as determined by ELISA . Symbols represent individual mice; horizontal lines show the mean. J , Serum IL ‐33 levels, determined by ELISA , in knockout, WT , and transgenic mice left untreated or treated with R848 (n = 10 per group). In B , C , E , G , and J , bars show the mean ± SD . * = P
    Figure Legend Snippet: Effects of TLR ‐7 agonist R848 in transgenic mice. A , Detection of GFP in bone marrow–derived macrophages ( BMM s) from mTLR ‐7−/− (knockout [ KO ]) mice and transgenic (Tg) mice by flow cytometric analysis, after staining with anti‐ CD 11b antibody. At least 2 independent experiments were performed. B , Interleukin‐6 ( IL ‐6) and IL ‐12p40 levels in BMM s from knockout mice and transgenic mice left unstimulated or stimulated with R848. After 24 hours of incubation, IL ‐6 and IL ‐12p40 were detected in culture medium by enzyme‐linked immunosorbent assay ( ELISA ). Results are from triplicate wells. At least 2 independent experiments were performed. C , Weight of SMG s, kidneys, lungs, and liver in 8‐week‐old knockout, wild‐type ( WT ), and transgenic mice treated with topical R848 for 4 weeks (n = 8 per group). D and F , H E‐stained ( D ) and Masson's trichrome–stained ( F ) sections of SMG s, pancreas, kidneys, lungs, and liver from representative 8‐week‐old R848‐treated knockout, WT , and transgenic mice. Bars = 100 μm. E and G , Focus score ( E ) and fibrosis score ( G ) for each organ in 8‐week‐old knockout, WT , and transgenic mice left untreated or treated with R848 (n = 8 per group). The fibrosis score was calculated from Masson's trichrome staining as described in Patients and Methods. HPF = high‐power field. H , Serial sections of SMG s from a representative 8‐week‐old R848‐treated transgenic mouse, stained with H E and for IgG1, TLR ‐7, CD 206, CD 317, and IL ‐33. Mayer's hematoxylin (blue) counterstained; bars = 100 μm. I , Serum IgG, IgG1, and IgG2a levels in knockout, WT , and transgenic mice (n = 10 per group) before and after R848 treatment, as determined by ELISA . Symbols represent individual mice; horizontal lines show the mean. J , Serum IL ‐33 levels, determined by ELISA , in knockout, WT , and transgenic mice left untreated or treated with R848 (n = 10 per group). In B , C , E , G , and J , bars show the mean ± SD . * = P

    Techniques Used: Transgenic Assay, Mouse Assay, Derivative Assay, Knock-Out, Staining, Incubation, Enzyme-linked Immunosorbent Assay

    Schematic model of the role of Toll‐like receptor 7 ( TLR ‐7)–positive M2 macrophages in the initiation of Ig4‐related disease. TLR ‐7 expressed on M2 macrophages recognize some RNA viruses or self RNA from apoptotic cells. Activated M2 macrophages secrete interleukin‐33 ( IL ‐33) and promote production of Th2 cytokines that lead to IgG4 class‐switching and fibrosis. TGF β = transforming growth factor β.
    Figure Legend Snippet: Schematic model of the role of Toll‐like receptor 7 ( TLR ‐7)–positive M2 macrophages in the initiation of Ig4‐related disease. TLR ‐7 expressed on M2 macrophages recognize some RNA viruses or self RNA from apoptotic cells. Activated M2 macrophages secrete interleukin‐33 ( IL ‐33) and promote production of Th2 cytokines that lead to IgG4 class‐switching and fibrosis. TGF β = transforming growth factor β.

    Techniques Used:

    Pathologic and serologic findings in human TLR ‐7–transgenic/mouse TLR ‐7–deficient (hu TLR ‐7–transgenic/ mTLR ‐7 −/− ) mice on a C57 BL /6 background. A , A wild‐type ( WT ) C57 BL /6 mouse and an hu TLR ‐7–transgenic/ mTLR ‐7−/− (transgenic [Tg]) mouse at 4 weeks old. B , Weight of SMG s, kidneys, lungs, and liver in 4‐week‐old WT mice (n = 5) and transgenic mice (n = 5). Bars show the mean ± SD . C , H E‐stained sections of SMG s, pancreas, kidneys, lungs, and liver from representative WT and transgenic mice. Bars = 100 μm. D , Focus score for each organ in WT mice (n = 5) and transgenic mice (n = 5). The focus score was estimated as described in Patients and Methods. Bars show the mean ± SD . HPF = high‐power field. E , Masson's trichrome–stained sections of SMG s, pancreas, kidneys, lungs, and liver from representative WT and transgenic mice. Masson's trichrome was used to stain nuclei (purple), cytoplasm (red), and collagen (connective or fibrotic tissue; blue) Bars = 100 μm. F , Fibrosis score for each organ in WT mice (n = 5) and transgenic mice (n = 5). The fibrosis score was calculated from Masson's trichrome staining as described in Patients and Methods. Bars show the mean ± SD . G , Serum IgG, IgG1, and IgG2a levels in WT mice (n = 10) and transgenic mice (n = 10), as determined by enzyme‐linked immunosorbent assay. Symbols represent individual mice; horizontal lines show the mean. * = P
    Figure Legend Snippet: Pathologic and serologic findings in human TLR ‐7–transgenic/mouse TLR ‐7–deficient (hu TLR ‐7–transgenic/ mTLR ‐7 −/− ) mice on a C57 BL /6 background. A , A wild‐type ( WT ) C57 BL /6 mouse and an hu TLR ‐7–transgenic/ mTLR ‐7−/− (transgenic [Tg]) mouse at 4 weeks old. B , Weight of SMG s, kidneys, lungs, and liver in 4‐week‐old WT mice (n = 5) and transgenic mice (n = 5). Bars show the mean ± SD . C , H E‐stained sections of SMG s, pancreas, kidneys, lungs, and liver from representative WT and transgenic mice. Bars = 100 μm. D , Focus score for each organ in WT mice (n = 5) and transgenic mice (n = 5). The focus score was estimated as described in Patients and Methods. Bars show the mean ± SD . HPF = high‐power field. E , Masson's trichrome–stained sections of SMG s, pancreas, kidneys, lungs, and liver from representative WT and transgenic mice. Masson's trichrome was used to stain nuclei (purple), cytoplasm (red), and collagen (connective or fibrotic tissue; blue) Bars = 100 μm. F , Fibrosis score for each organ in WT mice (n = 5) and transgenic mice (n = 5). The fibrosis score was calculated from Masson's trichrome staining as described in Patients and Methods. Bars show the mean ± SD . G , Serum IgG, IgG1, and IgG2a levels in WT mice (n = 10) and transgenic mice (n = 10), as determined by enzyme‐linked immunosorbent assay. Symbols represent individual mice; horizontal lines show the mean. * = P

    Techniques Used: Transgenic Assay, Mouse Assay, Staining, Enzyme-linked Immunosorbent Assay

    2) Product Images from "FoxP3 and Ezh2 regulate Tfr cell suppressive function and transcriptional program"

    Article Title: FoxP3 and Ezh2 regulate Tfr cell suppressive function and transcriptional program

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20181134

    FoxP3 instability in Tfr cells results in loss of suppressive function. (A) Identification of Tfr cells that down-regulate the transcription factor FoxP3. Gating strategy of ex-Tfr cells by flow cytometry from Foxp3 Cre Rosa26 Lox-stop-Lox-TdTomato (FoxP3 fate mapper) mice immunized with NP-OVA 7 d previously (left). Quantification of FoxP3 + cells as a frequency of CD4 + CXCR5 + TdTomato + cells (right). (B) Quantification of FoxP3 + cells as a frequency of CD4 + CXCR5 + TdTomato + cells from Foxp3 ERT2- Cre Rosa26 Lox-stop-Lox-TdTomato (inducible FoxP3 fate mapper) mice immunized with NP-OVA and injected with tamoxifen 7 d previously. (C) Expression of ICOS, CD25, GITR, and CTLA-4 on all CD4 + T cells, Tfr cells, or ex-Tfr cells from FoxP3 fate mapper mice as in A. (D) Expression of ICOS, CD25, GITR, and CTLA-4 on all CD4 + T cells, Tfr cells, or ex-Tfr cells from inducible FoxP3 fate mapper mice as in B. (E) Ex-Tfr cells fail to suppress Tfh-mediated B cell responses. In vitro suppression assay in which B and Tfh cells from WT mice were cultured with or without Tfr or ex-Tfr cells sorted from FoxP3 fate mapper mice as in A in the presence of anti-CD3/IgM for 6 d. IgG1 + GL7 + class-switched B cells are gated. Plots are pregated on CD19 + IA + CD4 − cells. (F) Quantification of Glut1 expression on B cells from suppression assays (Supp) as in E. (G) Quantification of IL-4 levels in culture supernatants from suppression assays as in E. (H) In vitro suppression assay in which B and Tfh cells from WT mice were cultured with or without Tfr or ex-Tfr cells sorted from inducible FoxP3 fate mapper mice as in B in the presence of anti-CD3/IgM for 6 d. IgG1 + GL7 + class-switched B cells are gated. Plots are pregated on CD19 + IA + CD4 − cells. (I) Quantification of Glut1 expression on B cells from suppression assays as in H. (J) Quantification of IL-4 levels in culture supernatants from suppression assays as in H. (K) Analysis of ex-Tfr cells after adoptive transfer of CD45.1 + Tfr cells (along with CD45.2 + Tfh cells) to Tcra −/− recipients which were immunized with NP-OVA and harvested 6 d later. Identification of ex-Tfr cells (left) and expression of CD25 and ICOS (right) are shown. All error bars indicate standard error. **, P
    Figure Legend Snippet: FoxP3 instability in Tfr cells results in loss of suppressive function. (A) Identification of Tfr cells that down-regulate the transcription factor FoxP3. Gating strategy of ex-Tfr cells by flow cytometry from Foxp3 Cre Rosa26 Lox-stop-Lox-TdTomato (FoxP3 fate mapper) mice immunized with NP-OVA 7 d previously (left). Quantification of FoxP3 + cells as a frequency of CD4 + CXCR5 + TdTomato + cells (right). (B) Quantification of FoxP3 + cells as a frequency of CD4 + CXCR5 + TdTomato + cells from Foxp3 ERT2- Cre Rosa26 Lox-stop-Lox-TdTomato (inducible FoxP3 fate mapper) mice immunized with NP-OVA and injected with tamoxifen 7 d previously. (C) Expression of ICOS, CD25, GITR, and CTLA-4 on all CD4 + T cells, Tfr cells, or ex-Tfr cells from FoxP3 fate mapper mice as in A. (D) Expression of ICOS, CD25, GITR, and CTLA-4 on all CD4 + T cells, Tfr cells, or ex-Tfr cells from inducible FoxP3 fate mapper mice as in B. (E) Ex-Tfr cells fail to suppress Tfh-mediated B cell responses. In vitro suppression assay in which B and Tfh cells from WT mice were cultured with or without Tfr or ex-Tfr cells sorted from FoxP3 fate mapper mice as in A in the presence of anti-CD3/IgM for 6 d. IgG1 + GL7 + class-switched B cells are gated. Plots are pregated on CD19 + IA + CD4 − cells. (F) Quantification of Glut1 expression on B cells from suppression assays (Supp) as in E. (G) Quantification of IL-4 levels in culture supernatants from suppression assays as in E. (H) In vitro suppression assay in which B and Tfh cells from WT mice were cultured with or without Tfr or ex-Tfr cells sorted from inducible FoxP3 fate mapper mice as in B in the presence of anti-CD3/IgM for 6 d. IgG1 + GL7 + class-switched B cells are gated. Plots are pregated on CD19 + IA + CD4 − cells. (I) Quantification of Glut1 expression on B cells from suppression assays as in H. (J) Quantification of IL-4 levels in culture supernatants from suppression assays as in H. (K) Analysis of ex-Tfr cells after adoptive transfer of CD45.1 + Tfr cells (along with CD45.2 + Tfh cells) to Tcra −/− recipients which were immunized with NP-OVA and harvested 6 d later. Identification of ex-Tfr cells (left) and expression of CD25 and ICOS (right) are shown. All error bars indicate standard error. **, P

    Techniques Used: Flow Cytometry, Cytometry, Mouse Assay, Injection, Expressing, In Vitro, Suppression Assay, Cell Culture, Adoptive Transfer Assay

    FoxP3 is sufficient to convert a Tfh program to a Tfr program. (A) Schematic of in vitro assay to express FoxP3 in Tfh cells. Control Thy1.1 retrovirus or FoxP3-encoding Thy1.1 retrovirus was added to cultures (as described in Materials and methods) along with anti-CD3/IgM for 4 d. (B) Representative gating of Thy1.1 + (transduced) and FoxP3-expressing Tfh cells from cultures as in A (left) and quantification of FoxP3 + Tfh cells reported as a percentage of Thy1.1 + transduced cells (right). Cells are pregated on CD4 + IA − cells. (C) Expression of CD25 on Thy1.1 + Tfh cells from experiments as in A. (D) CTLA-4 expression on Thy1.1 + Tfh cells from experiments as in A. (E and F) FoxP3 expression in Tfh cells results in suppression of B cell responses. Analysis of IgG1 + GL7 + class-switched B cells from cultures as in A. Representative gating (E) and quantification (F) are shown. (G) Glut1 expression in B cells from cultures as in A. (H) IL-4 levels in culture supernatants as in A. (I) Tfr-like FoxP3 + Tfh cells suppress nontransduced Tfh cells. Ki67 expression in Thy1.1 − Tfh cells from cultures as in A. Representative gating (left) and quantification (right) are shown. (J) Relative count of responder CD45.1 + Tfh cells which were added to cultures as in A 2 d after removal of virus and cultured for an additional 3 d. All error bars indicate standard error. *, P
    Figure Legend Snippet: FoxP3 is sufficient to convert a Tfh program to a Tfr program. (A) Schematic of in vitro assay to express FoxP3 in Tfh cells. Control Thy1.1 retrovirus or FoxP3-encoding Thy1.1 retrovirus was added to cultures (as described in Materials and methods) along with anti-CD3/IgM for 4 d. (B) Representative gating of Thy1.1 + (transduced) and FoxP3-expressing Tfh cells from cultures as in A (left) and quantification of FoxP3 + Tfh cells reported as a percentage of Thy1.1 + transduced cells (right). Cells are pregated on CD4 + IA − cells. (C) Expression of CD25 on Thy1.1 + Tfh cells from experiments as in A. (D) CTLA-4 expression on Thy1.1 + Tfh cells from experiments as in A. (E and F) FoxP3 expression in Tfh cells results in suppression of B cell responses. Analysis of IgG1 + GL7 + class-switched B cells from cultures as in A. Representative gating (E) and quantification (F) are shown. (G) Glut1 expression in B cells from cultures as in A. (H) IL-4 levels in culture supernatants as in A. (I) Tfr-like FoxP3 + Tfh cells suppress nontransduced Tfh cells. Ki67 expression in Thy1.1 − Tfh cells from cultures as in A. Representative gating (left) and quantification (right) are shown. (J) Relative count of responder CD45.1 + Tfh cells which were added to cultures as in A 2 d after removal of virus and cultured for an additional 3 d. All error bars indicate standard error. *, P

    Techniques Used: In Vitro, Expressing, Cell Culture

    Ezh2 is required for Tfr suppressive function and transcriptional program. (A) Conditional deletion of Ezh2 on T reg cells. Ezh2 f/f FoxP3 Cre/Cre or control ( Ezh2 f/+ FoxP3 Cre/Cre ) mice were immunized with NP-OVA, and 7 d later, T reg and Tfr cells were analyzed for expression of Ezh2 by flow cytometry. Representative gating (left) and quantification (right) are shown. MFI, mean fluorescence intensity. (B) Loss of Ezh2 results in increased Tfr cell percentages. Mice as in A were analyzed for Tfr cells. Representative gating (left) and quantification (middle, right) are shown. (C) Ezh2-deficient Tfr cells are less suppressive. In vitro suppression assay in which B and Tfh cells were cultured alone or along with Ezh2-sufficient or -deficient cells sorted as in B. IgG1 + GL7 + class-switched B cells gating (left), IgG1 + GL7 + class switched B cell quantification (middle), and quantification of Glut1 expression on B cells (right) are shown. (D) Ezh2 deficiency leads to a cell-intrinsic loss of Tfr cell suppressive function. Tfr cells from Ezh2 f/f FoxP3 Cre/+ or Ezh2 f/+ FoxP3 Cre/+ mice (sorted based on Cre-YFP) were used for suppression assays as in C. Representative flow cytometry of IgG1 + GL7 + class-switched B cells (left) and IgG secretion (right) are shown. (E) Loss of Ezh2 results in loss of the Tfr cell transcriptional program. PCA of RNA-seq transcriptional data from Ezh2-deficient ( Ezh2 f/f FoxP3 Cre ) or Ezh2-sufficient ( Ezh2 f/+ FoxP3 Cre ) Tfr and T reg cells sorted as in B. (F) Volcano plots showing control or Ezh2-deficient Tfr cell RNA-seq data with total T reg genes (T reg versus T conv; left), Tfr (follicular) genes (Tfr versus Tfh; middle), and Tfr (T reg) genes (Tfr versus T reg; right; from Fig. 1 ; in red) compared with all genes (gray). P value was calculated using a χ 2 test. (G) Enrichment score traces from GSEA analysis comparing Ezh2 F/+ Tfr cells versus F/+ T reg or F/F Tfr cells using indicated gene sets (generated from data in Fig. 1 ). (H and I) Venn diagram illustrating the overlap of genes differentially expressed in Ezh2-deficient Tfr cells versus control Tfr cells (ΔEzh2) compared with either genes differentially expressed in FoxP3-deleted Tfr cells compared with control Tfr cells (ΔFoxP3) or genes differentially expressed in in vitro generated ex-Tfr versus Tfr cells (FoxP3 Down). All error bars indicate standard error. **, P
    Figure Legend Snippet: Ezh2 is required for Tfr suppressive function and transcriptional program. (A) Conditional deletion of Ezh2 on T reg cells. Ezh2 f/f FoxP3 Cre/Cre or control ( Ezh2 f/+ FoxP3 Cre/Cre ) mice were immunized with NP-OVA, and 7 d later, T reg and Tfr cells were analyzed for expression of Ezh2 by flow cytometry. Representative gating (left) and quantification (right) are shown. MFI, mean fluorescence intensity. (B) Loss of Ezh2 results in increased Tfr cell percentages. Mice as in A were analyzed for Tfr cells. Representative gating (left) and quantification (middle, right) are shown. (C) Ezh2-deficient Tfr cells are less suppressive. In vitro suppression assay in which B and Tfh cells were cultured alone or along with Ezh2-sufficient or -deficient cells sorted as in B. IgG1 + GL7 + class-switched B cells gating (left), IgG1 + GL7 + class switched B cell quantification (middle), and quantification of Glut1 expression on B cells (right) are shown. (D) Ezh2 deficiency leads to a cell-intrinsic loss of Tfr cell suppressive function. Tfr cells from Ezh2 f/f FoxP3 Cre/+ or Ezh2 f/+ FoxP3 Cre/+ mice (sorted based on Cre-YFP) were used for suppression assays as in C. Representative flow cytometry of IgG1 + GL7 + class-switched B cells (left) and IgG secretion (right) are shown. (E) Loss of Ezh2 results in loss of the Tfr cell transcriptional program. PCA of RNA-seq transcriptional data from Ezh2-deficient ( Ezh2 f/f FoxP3 Cre ) or Ezh2-sufficient ( Ezh2 f/+ FoxP3 Cre ) Tfr and T reg cells sorted as in B. (F) Volcano plots showing control or Ezh2-deficient Tfr cell RNA-seq data with total T reg genes (T reg versus T conv; left), Tfr (follicular) genes (Tfr versus Tfh; middle), and Tfr (T reg) genes (Tfr versus T reg; right; from Fig. 1 ; in red) compared with all genes (gray). P value was calculated using a χ 2 test. (G) Enrichment score traces from GSEA analysis comparing Ezh2 F/+ Tfr cells versus F/+ T reg or F/F Tfr cells using indicated gene sets (generated from data in Fig. 1 ). (H and I) Venn diagram illustrating the overlap of genes differentially expressed in Ezh2-deficient Tfr cells versus control Tfr cells (ΔEzh2) compared with either genes differentially expressed in FoxP3-deleted Tfr cells compared with control Tfr cells (ΔFoxP3) or genes differentially expressed in in vitro generated ex-Tfr versus Tfr cells (FoxP3 Down). All error bars indicate standard error. **, P

    Techniques Used: Mouse Assay, Expressing, Flow Cytometry, Cytometry, Fluorescence, In Vitro, Suppression Assay, Cell Culture, RNA Sequencing Assay, Generated

    3) Product Images from "Screening for IgG4-type anti-nuclear antibodies in IgG4-related disease"

    Article Title: Screening for IgG4-type anti-nuclear antibodies in IgG4-related disease

    Journal: BMC Musculoskeletal Disorders

    doi: 10.1186/s12891-015-0584-4

    Subclass-based ANA test of a patient with Sjögren’s syndrome (SS #4) showing IgG4-type ANA. ANA patterns differed among IgG subclasses. Total IgG showed Speckled, while IgG2 showed Speckled + Cyto, IgG1 and IgG3 showed Nucleolar + Cyto (with atypical cytoplasmic spots), and IgG4 showed Peripheral. Bar = 20 μm. Cyto: cytoplasmic
    Figure Legend Snippet: Subclass-based ANA test of a patient with Sjögren’s syndrome (SS #4) showing IgG4-type ANA. ANA patterns differed among IgG subclasses. Total IgG showed Speckled, while IgG2 showed Speckled + Cyto, IgG1 and IgG3 showed Nucleolar + Cyto (with atypical cytoplasmic spots), and IgG4 showed Peripheral. Bar = 20 μm. Cyto: cytoplasmic

    Techniques Used:

    Subclass-based ANA test for systemic autoimmune diseases showing immunofluorescence microscopy for each typical case, including systemic lupus erythematosus (SLE #1), systemic sclerosis (SSc #2), Sjögren’s syndrome (SS #8) and polymyositis (PM #2) showed variation in ANA patterns among IgG subclasses. In SSc #2, total IgG showed Discrete spe + Speckled + Cyto, while IgG1 showed Discrete spe + Speckled, IgG2 showed Discrete spe + Speckled + Cyto, IgG3 showed Discrete spe + Cyto, and IgG4 showed negative. In SS #8, total IgG showed Speckled + Nucleolar + Cyto, while IgG1 and IgG2 showed Speckled + Cyto, IgG3 showed Nucleolar + Cyto, and IgG4 showed negative. Bar = 20 μm Discrete spe: discrete speckled, Cyto: cytoplasmic
    Figure Legend Snippet: Subclass-based ANA test for systemic autoimmune diseases showing immunofluorescence microscopy for each typical case, including systemic lupus erythematosus (SLE #1), systemic sclerosis (SSc #2), Sjögren’s syndrome (SS #8) and polymyositis (PM #2) showed variation in ANA patterns among IgG subclasses. In SSc #2, total IgG showed Discrete spe + Speckled + Cyto, while IgG1 showed Discrete spe + Speckled, IgG2 showed Discrete spe + Speckled + Cyto, IgG3 showed Discrete spe + Cyto, and IgG4 showed negative. In SS #8, total IgG showed Speckled + Nucleolar + Cyto, while IgG1 and IgG2 showed Speckled + Cyto, IgG3 showed Nucleolar + Cyto, and IgG4 showed negative. Bar = 20 μm Discrete spe: discrete speckled, Cyto: cytoplasmic

    Techniques Used: Immunofluorescence, Microscopy

    Subclass-based ANA test in IgG4-RD, showing immunofluorescence microscopy of two typical IgG4-RD cases (IgG4-RD #3 and #5). Lower right panel: We confirmed the second antibody’s function by direct immunofluorescence of a lymph node specimen (IgG4 + /IgG + plasma cell ratio = 0.69) from an IgG4-RD patient (IgG4-RD #14). Bar = 20 μm
    Figure Legend Snippet: Subclass-based ANA test in IgG4-RD, showing immunofluorescence microscopy of two typical IgG4-RD cases (IgG4-RD #3 and #5). Lower right panel: We confirmed the second antibody’s function by direct immunofluorescence of a lymph node specimen (IgG4 + /IgG + plasma cell ratio = 0.69) from an IgG4-RD patient (IgG4-RD #14). Bar = 20 μm

    Techniques Used: Immunofluorescence, Microscopy

    4) Product Images from "Screening for IgG4-type anti-nuclear antibodies in IgG4-related disease"

    Article Title: Screening for IgG4-type anti-nuclear antibodies in IgG4-related disease

    Journal: BMC Musculoskeletal Disorders

    doi: 10.1186/s12891-015-0584-4

    Subclass-based ANA test of a patient with Sjögren’s syndrome (SS #4) showing IgG4-type ANA. ANA patterns differed among IgG subclasses. Total IgG showed Speckled, while IgG2 showed Speckled + Cyto, IgG1 and IgG3 showed Nucleolar + Cyto (with atypical cytoplasmic spots), and IgG4 showed Peripheral. Bar = 20 μm. Cyto: cytoplasmic
    Figure Legend Snippet: Subclass-based ANA test of a patient with Sjögren’s syndrome (SS #4) showing IgG4-type ANA. ANA patterns differed among IgG subclasses. Total IgG showed Speckled, while IgG2 showed Speckled + Cyto, IgG1 and IgG3 showed Nucleolar + Cyto (with atypical cytoplasmic spots), and IgG4 showed Peripheral. Bar = 20 μm. Cyto: cytoplasmic

    Techniques Used:

    Subclass-based ANA test for systemic autoimmune diseases showing immunofluorescence microscopy for each typical case, including systemic lupus erythematosus (SLE #1), systemic sclerosis (SSc #2), Sjögren’s syndrome (SS #8) and polymyositis (PM #2) showed variation in ANA patterns among IgG subclasses. In SSc #2, total IgG showed Discrete spe + Speckled + Cyto, while IgG1 showed Discrete spe + Speckled, IgG2 showed Discrete spe + Speckled + Cyto, IgG3 showed Discrete spe + Cyto, and IgG4 showed negative. In SS #8, total IgG showed Speckled + Nucleolar + Cyto, while IgG1 and IgG2 showed Speckled + Cyto, IgG3 showed Nucleolar + Cyto, and IgG4 showed negative. Bar = 20 μm Discrete spe: discrete speckled, Cyto: cytoplasmic
    Figure Legend Snippet: Subclass-based ANA test for systemic autoimmune diseases showing immunofluorescence microscopy for each typical case, including systemic lupus erythematosus (SLE #1), systemic sclerosis (SSc #2), Sjögren’s syndrome (SS #8) and polymyositis (PM #2) showed variation in ANA patterns among IgG subclasses. In SSc #2, total IgG showed Discrete spe + Speckled + Cyto, while IgG1 showed Discrete spe + Speckled, IgG2 showed Discrete spe + Speckled + Cyto, IgG3 showed Discrete spe + Cyto, and IgG4 showed negative. In SS #8, total IgG showed Speckled + Nucleolar + Cyto, while IgG1 and IgG2 showed Speckled + Cyto, IgG3 showed Nucleolar + Cyto, and IgG4 showed negative. Bar = 20 μm Discrete spe: discrete speckled, Cyto: cytoplasmic

    Techniques Used: Immunofluorescence, Microscopy

    Subclass-based ANA test in IgG4-RD, showing immunofluorescence microscopy of two typical IgG4-RD cases (IgG4-RD #3 and #5). Lower right panel: We confirmed the second antibody’s function by direct immunofluorescence of a lymph node specimen (IgG4 + /IgG + plasma cell ratio = 0.69) from an IgG4-RD patient (IgG4-RD #14). Bar = 20 μm
    Figure Legend Snippet: Subclass-based ANA test in IgG4-RD, showing immunofluorescence microscopy of two typical IgG4-RD cases (IgG4-RD #3 and #5). Lower right panel: We confirmed the second antibody’s function by direct immunofluorescence of a lymph node specimen (IgG4 + /IgG + plasma cell ratio = 0.69) from an IgG4-RD patient (IgG4-RD #14). Bar = 20 μm

    Techniques Used: Immunofluorescence, Microscopy

    5) Product Images from "FoxP3 and Ezh2 regulate Tfr cell suppressive function and transcriptional program"

    Article Title: FoxP3 and Ezh2 regulate Tfr cell suppressive function and transcriptional program

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20181134

    FoxP3 instability in Tfr cells results in loss of suppressive function. (A) Identification of Tfr cells that down-regulate the transcription factor FoxP3. Gating strategy of ex-Tfr cells by flow cytometry from Foxp3 Cre Rosa26 Lox-stop-Lox-TdTomato (FoxP3 fate mapper) mice immunized with NP-OVA 7 d previously (left). Quantification of FoxP3 + cells as a frequency of CD4 + CXCR5 + TdTomato + cells (right). (B) Quantification of FoxP3 + cells as a frequency of CD4 + CXCR5 + TdTomato + cells from Foxp3 ERT2- Cre Rosa26 Lox-stop-Lox-TdTomato (inducible FoxP3 fate mapper) mice immunized with NP-OVA and injected with tamoxifen 7 d previously. (C) Expression of ICOS, CD25, GITR, and CTLA-4 on all CD4 + T cells, Tfr cells, or ex-Tfr cells from FoxP3 fate mapper mice as in A. (D) Expression of ICOS, CD25, GITR, and CTLA-4 on all CD4 + T cells, Tfr cells, or ex-Tfr cells from inducible FoxP3 fate mapper mice as in B. (E) Ex-Tfr cells fail to suppress Tfh-mediated B cell responses. In vitro suppression assay in which B and Tfh cells from WT mice were cultured with or without Tfr or ex-Tfr cells sorted from FoxP3 fate mapper mice as in A in the presence of anti-CD3/IgM for 6 d. IgG1 + GL7 + class-switched B cells are gated. Plots are pregated on CD19 + IA + CD4 − cells. (F) Quantification of Glut1 expression on B cells from suppression assays (Supp) as in E. (G) Quantification of IL-4 levels in culture supernatants from suppression assays as in E. (H) In vitro suppression assay in which B and Tfh cells from WT mice were cultured with or without Tfr or ex-Tfr cells sorted from inducible FoxP3 fate mapper mice as in B in the presence of anti-CD3/IgM for 6 d. IgG1 + GL7 + class-switched B cells are gated. Plots are pregated on CD19 + IA + CD4 − cells. (I) Quantification of Glut1 expression on B cells from suppression assays as in H. (J) Quantification of IL-4 levels in culture supernatants from suppression assays as in H. (K) Analysis of ex-Tfr cells after adoptive transfer of CD45.1 + Tfr cells (along with CD45.2 + Tfh cells) to Tcra −/− recipients which were immunized with NP-OVA and harvested 6 d later. Identification of ex-Tfr cells (left) and expression of CD25 and ICOS (right) are shown. All error bars indicate standard error. **, P
    Figure Legend Snippet: FoxP3 instability in Tfr cells results in loss of suppressive function. (A) Identification of Tfr cells that down-regulate the transcription factor FoxP3. Gating strategy of ex-Tfr cells by flow cytometry from Foxp3 Cre Rosa26 Lox-stop-Lox-TdTomato (FoxP3 fate mapper) mice immunized with NP-OVA 7 d previously (left). Quantification of FoxP3 + cells as a frequency of CD4 + CXCR5 + TdTomato + cells (right). (B) Quantification of FoxP3 + cells as a frequency of CD4 + CXCR5 + TdTomato + cells from Foxp3 ERT2- Cre Rosa26 Lox-stop-Lox-TdTomato (inducible FoxP3 fate mapper) mice immunized with NP-OVA and injected with tamoxifen 7 d previously. (C) Expression of ICOS, CD25, GITR, and CTLA-4 on all CD4 + T cells, Tfr cells, or ex-Tfr cells from FoxP3 fate mapper mice as in A. (D) Expression of ICOS, CD25, GITR, and CTLA-4 on all CD4 + T cells, Tfr cells, or ex-Tfr cells from inducible FoxP3 fate mapper mice as in B. (E) Ex-Tfr cells fail to suppress Tfh-mediated B cell responses. In vitro suppression assay in which B and Tfh cells from WT mice were cultured with or without Tfr or ex-Tfr cells sorted from FoxP3 fate mapper mice as in A in the presence of anti-CD3/IgM for 6 d. IgG1 + GL7 + class-switched B cells are gated. Plots are pregated on CD19 + IA + CD4 − cells. (F) Quantification of Glut1 expression on B cells from suppression assays (Supp) as in E. (G) Quantification of IL-4 levels in culture supernatants from suppression assays as in E. (H) In vitro suppression assay in which B and Tfh cells from WT mice were cultured with or without Tfr or ex-Tfr cells sorted from inducible FoxP3 fate mapper mice as in B in the presence of anti-CD3/IgM for 6 d. IgG1 + GL7 + class-switched B cells are gated. Plots are pregated on CD19 + IA + CD4 − cells. (I) Quantification of Glut1 expression on B cells from suppression assays as in H. (J) Quantification of IL-4 levels in culture supernatants from suppression assays as in H. (K) Analysis of ex-Tfr cells after adoptive transfer of CD45.1 + Tfr cells (along with CD45.2 + Tfh cells) to Tcra −/− recipients which were immunized with NP-OVA and harvested 6 d later. Identification of ex-Tfr cells (left) and expression of CD25 and ICOS (right) are shown. All error bars indicate standard error. **, P

    Techniques Used: Flow Cytometry, Cytometry, Mouse Assay, Injection, Expressing, In Vitro, Suppression Assay, Cell Culture, IA, Adoptive Transfer Assay

    FoxP3 is sufficient to convert a Tfh program to a Tfr program. (A) Schematic of in vitro assay to express FoxP3 in Tfh cells. Control Thy1.1 retrovirus or FoxP3-encoding Thy1.1 retrovirus was added to cultures (as described in Materials and methods) along with anti-CD3/IgM for 4 d. (B) Representative gating of Thy1.1 + (transduced) and FoxP3-expressing Tfh cells from cultures as in A (left) and quantification of FoxP3 + Tfh cells reported as a percentage of Thy1.1 + transduced cells (right). Cells are pregated on CD4 + IA − cells. (C) Expression of CD25 on Thy1.1 + Tfh cells from experiments as in A. (D) CTLA-4 expression on Thy1.1 + Tfh cells from experiments as in A. (E and F) FoxP3 expression in Tfh cells results in suppression of B cell responses. Analysis of IgG1 + GL7 + class-switched B cells from cultures as in A. Representative gating (E) and quantification (F) are shown. (G) Glut1 expression in B cells from cultures as in A. (H) IL-4 levels in culture supernatants as in A. (I) Tfr-like FoxP3 + Tfh cells suppress nontransduced Tfh cells. Ki67 expression in Thy1.1 − Tfh cells from cultures as in A. Representative gating (left) and quantification (right) are shown. (J) Relative count of responder CD45.1 + Tfh cells which were added to cultures as in A 2 d after removal of virus and cultured for an additional 3 d. All error bars indicate standard error. *, P
    Figure Legend Snippet: FoxP3 is sufficient to convert a Tfh program to a Tfr program. (A) Schematic of in vitro assay to express FoxP3 in Tfh cells. Control Thy1.1 retrovirus or FoxP3-encoding Thy1.1 retrovirus was added to cultures (as described in Materials and methods) along with anti-CD3/IgM for 4 d. (B) Representative gating of Thy1.1 + (transduced) and FoxP3-expressing Tfh cells from cultures as in A (left) and quantification of FoxP3 + Tfh cells reported as a percentage of Thy1.1 + transduced cells (right). Cells are pregated on CD4 + IA − cells. (C) Expression of CD25 on Thy1.1 + Tfh cells from experiments as in A. (D) CTLA-4 expression on Thy1.1 + Tfh cells from experiments as in A. (E and F) FoxP3 expression in Tfh cells results in suppression of B cell responses. Analysis of IgG1 + GL7 + class-switched B cells from cultures as in A. Representative gating (E) and quantification (F) are shown. (G) Glut1 expression in B cells from cultures as in A. (H) IL-4 levels in culture supernatants as in A. (I) Tfr-like FoxP3 + Tfh cells suppress nontransduced Tfh cells. Ki67 expression in Thy1.1 − Tfh cells from cultures as in A. Representative gating (left) and quantification (right) are shown. (J) Relative count of responder CD45.1 + Tfh cells which were added to cultures as in A 2 d after removal of virus and cultured for an additional 3 d. All error bars indicate standard error. *, P

    Techniques Used: In Vitro, Expressing, IA, Cell Culture

    Related Articles

    Staining:

    Article Title: FoxP3 and Ezh2 regulate Tfr cell suppressive function and transcriptional program
    Article Snippet: .. Samples were then intracellularly stained with anti-IgG1 (A85-1), anti-FoxP3 (FJK-16S), anti-Ki67 (B56), anti-Glut1 (polyclonal; Abcam), anti-Helios (22F6), or CTLA-4 (UC10-4B9). ..

    Incubation:

    Article Title: Evolution of the Immune Response against Recombinant Proteins (TcpA, TcpB, and FlaA) as a Candidate Subunit Cholera Vaccine
    Article Snippet: .. The plates were kept at 37°C for 2 h. After washing, 50 μ L of 1/10000 diluted antibodies against IgG1 and IgG2a conjugated classes was added to HRP (Abcam, UK) and they were kept at 37°C for 2 h. Finally, light absorption at wavelength of 490 nm was determined by adding 100 μ L of o-phenylenediamine (OPD) substrate solution to each well and incubation for 10 min. .. Proliferative Response Check by MTT Proliferation was checked by MTT method [ ].

    Article Title: Recognition of tumor cells by Dectin-1 orchestrates innate immune cells for anti-tumor responses
    Article Snippet: .. Flow cytometric assay Cells were incubated with sDectin-1 or human IgG1 Fc in TBS (pH 8.0) containing 1.3 mM CaCl2 for 15 min at 4°C, and then labeled with anti-human IgG1 antibody (4E3; Abcam, Cambridge, MA) conjugated with allophycocyanin (APC). .. The cells were then analyzed by LSRII/Fortessa (BD Biosciences).

    Chromatin Immunoprecipitation:

    Article Title: Oct4 regulates DNA methyltransferase 1 transcription by direct binding of the regulatory element
    Article Snippet: .. The antibodies used for ChIP studies were anti-RNA Pol II antibody, anti-IgG antibody (provided in the kit) and anti-Oct4 antibody (Abcam, ab19857). .. After reverse cross-linking and DNA purification, DNA from input (1:10 diluted) or immunoprecipitated samples was assayed via PCR, and the products were separated using 1.5% agarose gel electrophoresis.

    Flow Cytometry:

    Article Title: Recognition of tumor cells by Dectin-1 orchestrates innate immune cells for anti-tumor responses
    Article Snippet: .. Flow cytometric assay Cells were incubated with sDectin-1 or human IgG1 Fc in TBS (pH 8.0) containing 1.3 mM CaCl2 for 15 min at 4°C, and then labeled with anti-human IgG1 antibody (4E3; Abcam, Cambridge, MA) conjugated with allophycocyanin (APC). .. The cells were then analyzed by LSRII/Fortessa (BD Biosciences).

    Labeling:

    Article Title: Recognition of tumor cells by Dectin-1 orchestrates innate immune cells for anti-tumor responses
    Article Snippet: .. Flow cytometric assay Cells were incubated with sDectin-1 or human IgG1 Fc in TBS (pH 8.0) containing 1.3 mM CaCl2 for 15 min at 4°C, and then labeled with anti-human IgG1 antibody (4E3; Abcam, Cambridge, MA) conjugated with allophycocyanin (APC). .. The cells were then analyzed by LSRII/Fortessa (BD Biosciences).

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    Abcam goat anti mouse igg
    Internalization of WB‐6 into living human monocytic leukemia cell line THP‐1 cells and human umbilical vein endothelial cells (HUVECs). (a) THP‐1 cells were incubated with 25 µg/ml WB‐6, isotype‐matched control (IC) or 5 µg/ml 2C10 for 2 h. After washing, fixation, permeabilization and blocking, internalized immunoglobulin (Ig)G was detected using <t>Alexa</t> Fluor 488‐labeled goat anti‐mouse <t>IgG</t> (green). (b) THP‐1 cells were incubated with (center column) or without (left column) 25 µg/ml WB‐6 for 2 h or 1 µM staurosporine for 6 h (right column), and expression of phosphatidylserine was tested using annexin V‐biotin and fluorescein isothiocyanate (FITC)‐streptavidin (green). (c) THP‐1 cells were incubated with 25 µg/ml WB‐6 for 2 h, and Alexa Fluor 488‐labeled goat anti‐mouse IgG (green) was added after (left) or before (right) fixation and permeabilization. (d) Internalization of WB‐6 into HUVECs was tested according to the same protocol as (a). In all experiments, the nuclei were stained with Hoechst 33342 (blue). A representative of three independent experiments with similar results is shown.
    Goat Anti Mouse Igg, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 86 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Domain formation in B. burgdorferi as determined by immunogold TEM analysis requires raft-supporting sterols. A. Representative negative-stain TEM images of B. burgdorferi substituted with the indicated sterols and probed for sterol glycolipids using a rabbit antibody to <t>asialo-GM1</t> followed by a secondary anti-rabbit antibody conjugated to 6 nm colloidal gold. Micrographs show electron dense regions, which are portions of B. burgdorferi and show associated gold particles. Two images (of about 400 nm long segments) are shown for each sterol. Top row, sterols strongly supporting ordered domain formation; middle row, sterols with an intermediate ability to form ordered domains; bottom row, sterols that inhibit ordered domain formation [22] , [26] – [28] . Boxes highlight sterol glycolipid clusters. Bars = 100 nm. TEM micrographs for additional sterols are shown in Fig. S3 in Text S1 . B. Pooled “K-function” analysis of TEM experiments. The spatial distribution of gold particles is presented as curves representing the mean values of L(r)-r from images of three different bacteria from three independent sterol substitution experiments. Values of L(r)-r above the CI (95% confidence level, dashed line) indicate clustering (i.e. domain formation) of the sterol glycolipid at that specific length scale.
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    Microscopy images (×40) of immunofluorescent staining of <t>LAT-1</t> (tumour (a) , granuloma (d) ), GLUT-1 (tumour (b) , granuloma (e) ) and H E staining (tumour (c) , granuloma (f) ). White arrowhead : cancer cell; black arrow : lymphocyte infiltration; white arrow : epithelioid cell granuloma. Bars indicate 50 μm.
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    Abcam sheep anti mouse albumin polyclonal igg antibody
    Co-localization of GFP fluorescence and α-GFP immunofluorescence in tissue sections of cysticerci after 24 h of microinjection. a GFP fluorescence in a GFP-TOPO plasmid microinjected cyst (FITC filter); b Immunohistochemical localization using a 1:250 dilution of a <t>polyclonal</t> α -GFP rabbit <t>IgG</t> antibody followed by a 1:200 dilution of a goat α-rabbit IgG CY3-conjugated antibody (CY3 filter); c Merging of a and b images. d Water microinjected cysts (FITC filter); e Immunohistochemical localization as in b in a water microinjected cyst (CY3 filter); f Merging of d and e images. Arrows show subtegumentary cytons. T tegument. Photographs were obtained under Confocal Laser microscopy
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    Internalization of WB‐6 into living human monocytic leukemia cell line THP‐1 cells and human umbilical vein endothelial cells (HUVECs). (a) THP‐1 cells were incubated with 25 µg/ml WB‐6, isotype‐matched control (IC) or 5 µg/ml 2C10 for 2 h. After washing, fixation, permeabilization and blocking, internalized immunoglobulin (Ig)G was detected using Alexa Fluor 488‐labeled goat anti‐mouse IgG (green). (b) THP‐1 cells were incubated with (center column) or without (left column) 25 µg/ml WB‐6 for 2 h or 1 µM staurosporine for 6 h (right column), and expression of phosphatidylserine was tested using annexin V‐biotin and fluorescein isothiocyanate (FITC)‐streptavidin (green). (c) THP‐1 cells were incubated with 25 µg/ml WB‐6 for 2 h, and Alexa Fluor 488‐labeled goat anti‐mouse IgG (green) was added after (left) or before (right) fixation and permeabilization. (d) Internalization of WB‐6 into HUVECs was tested according to the same protocol as (a). In all experiments, the nuclei were stained with Hoechst 33342 (blue). A representative of three independent experiments with similar results is shown.

    Journal: Clinical and Experimental Immunology

    Article Title: Anti‐β2‐glycoprotein I antibody with DNA binding activity enters living monocytes via cell surface DNA and induces tissue factor expression

    doi: 10.1111/cei.13229

    Figure Lengend Snippet: Internalization of WB‐6 into living human monocytic leukemia cell line THP‐1 cells and human umbilical vein endothelial cells (HUVECs). (a) THP‐1 cells were incubated with 25 µg/ml WB‐6, isotype‐matched control (IC) or 5 µg/ml 2C10 for 2 h. After washing, fixation, permeabilization and blocking, internalized immunoglobulin (Ig)G was detected using Alexa Fluor 488‐labeled goat anti‐mouse IgG (green). (b) THP‐1 cells were incubated with (center column) or without (left column) 25 µg/ml WB‐6 for 2 h or 1 µM staurosporine for 6 h (right column), and expression of phosphatidylserine was tested using annexin V‐biotin and fluorescein isothiocyanate (FITC)‐streptavidin (green). (c) THP‐1 cells were incubated with 25 µg/ml WB‐6 for 2 h, and Alexa Fluor 488‐labeled goat anti‐mouse IgG (green) was added after (left) or before (right) fixation and permeabilization. (d) Internalization of WB‐6 into HUVECs was tested according to the same protocol as (a). In all experiments, the nuclei were stained with Hoechst 33342 (blue). A representative of three independent experiments with similar results is shown.

    Article Snippet: When we added Alexa Fluor 488‐labeled goat anti‐mouse IgG before fixation and permeabilization, virtually all cells remained unstained, as shown in Fig. c. These results again demonstrate the integrity of the cell membrane, because the second antibody could not enter the cells.

    Techniques: Western Blot, Incubation, Blocking Assay, Labeling, Expressing, Staining

    Internalization of WB‐6 into living peripheral blood mononuclear cells (PBMCs). (a) PBMCs from healthy volunteers were incubated with WB‐6 for 2 h and internalized antibody was detected as described in Fig. 2 . A representative immunofluorescence image showing that WB‐6 entered only a small fraction of PBMCs. (b) In flow cytometric analysis, forward‐ and side‐scatter plot of PBMCs showed two populations of the cells, designated P1 and P2, representing monocytes and lymphocytes, respectively. (c) Most of the cells in the P1 gate were confirmed to be CD14 + . (d–f) Representative histograms showing the ratio of Alexa Fluor 488‐positive cells after 2 h incubation of PBMCs with isotype‐matched IgG in the gate P1 (d), WB‐6 in the gate P1 (e) and WB‐6 in the gate P2 (f). (g) The ratio of Alexa Fluor 488‐positive cells in the gate P1 (Mono) and gate P2 (Lym). Data show a representative of three independent experiments with similar results, and the mean ± standard error of the mean (s.e.m.) of triplicate assay.

    Journal: Clinical and Experimental Immunology

    Article Title: Anti‐β2‐glycoprotein I antibody with DNA binding activity enters living monocytes via cell surface DNA and induces tissue factor expression

    doi: 10.1111/cei.13229

    Figure Lengend Snippet: Internalization of WB‐6 into living peripheral blood mononuclear cells (PBMCs). (a) PBMCs from healthy volunteers were incubated with WB‐6 for 2 h and internalized antibody was detected as described in Fig. 2 . A representative immunofluorescence image showing that WB‐6 entered only a small fraction of PBMCs. (b) In flow cytometric analysis, forward‐ and side‐scatter plot of PBMCs showed two populations of the cells, designated P1 and P2, representing monocytes and lymphocytes, respectively. (c) Most of the cells in the P1 gate were confirmed to be CD14 + . (d–f) Representative histograms showing the ratio of Alexa Fluor 488‐positive cells after 2 h incubation of PBMCs with isotype‐matched IgG in the gate P1 (d), WB‐6 in the gate P1 (e) and WB‐6 in the gate P2 (f). (g) The ratio of Alexa Fluor 488‐positive cells in the gate P1 (Mono) and gate P2 (Lym). Data show a representative of three independent experiments with similar results, and the mean ± standard error of the mean (s.e.m.) of triplicate assay.

    Article Snippet: When we added Alexa Fluor 488‐labeled goat anti‐mouse IgG before fixation and permeabilization, virtually all cells remained unstained, as shown in Fig. c. These results again demonstrate the integrity of the cell membrane, because the second antibody could not enter the cells.

    Techniques: Western Blot, Incubation, Immunofluorescence, Flow Cytometry

    Domain formation in B. burgdorferi as determined by immunogold TEM analysis requires raft-supporting sterols. A. Representative negative-stain TEM images of B. burgdorferi substituted with the indicated sterols and probed for sterol glycolipids using a rabbit antibody to asialo-GM1 followed by a secondary anti-rabbit antibody conjugated to 6 nm colloidal gold. Micrographs show electron dense regions, which are portions of B. burgdorferi and show associated gold particles. Two images (of about 400 nm long segments) are shown for each sterol. Top row, sterols strongly supporting ordered domain formation; middle row, sterols with an intermediate ability to form ordered domains; bottom row, sterols that inhibit ordered domain formation [22] , [26] – [28] . Boxes highlight sterol glycolipid clusters. Bars = 100 nm. TEM micrographs for additional sterols are shown in Fig. S3 in Text S1 . B. Pooled “K-function” analysis of TEM experiments. The spatial distribution of gold particles is presented as curves representing the mean values of L(r)-r from images of three different bacteria from three independent sterol substitution experiments. Values of L(r)-r above the CI (95% confidence level, dashed line) indicate clustering (i.e. domain formation) of the sterol glycolipid at that specific length scale.

    Journal: PLoS Pathogens

    Article Title: Proving Lipid Rafts Exist: Membrane Domains in the Prokaryote Borrelia burgdorferi Have the Same Properties as Eukaryotic Lipid Rafts

    doi: 10.1371/journal.ppat.1003353

    Figure Lengend Snippet: Domain formation in B. burgdorferi as determined by immunogold TEM analysis requires raft-supporting sterols. A. Representative negative-stain TEM images of B. burgdorferi substituted with the indicated sterols and probed for sterol glycolipids using a rabbit antibody to asialo-GM1 followed by a secondary anti-rabbit antibody conjugated to 6 nm colloidal gold. Micrographs show electron dense regions, which are portions of B. burgdorferi and show associated gold particles. Two images (of about 400 nm long segments) are shown for each sterol. Top row, sterols strongly supporting ordered domain formation; middle row, sterols with an intermediate ability to form ordered domains; bottom row, sterols that inhibit ordered domain formation [22] , [26] – [28] . Boxes highlight sterol glycolipid clusters. Bars = 100 nm. TEM micrographs for additional sterols are shown in Fig. S3 in Text S1 . B. Pooled “K-function” analysis of TEM experiments. The spatial distribution of gold particles is presented as curves representing the mean values of L(r)-r from images of three different bacteria from three independent sterol substitution experiments. Values of L(r)-r above the CI (95% confidence level, dashed line) indicate clustering (i.e. domain formation) of the sterol glycolipid at that specific length scale.

    Article Snippet: For slot blots, fractions were loaded onto nitrocellulose and probed with anti-asialo GM1 (for sterol glycolipids, rabbit polyclonal IgG, AbCam) or monoclonal antibodies CB2 (anti-OspB, mouse IgG) or CB10 (anti-OspA, mouse IgG) , .

    Techniques: Transmission Electron Microscopy, Staining

    Microscopy images (×40) of immunofluorescent staining of LAT-1 (tumour (a) , granuloma (d) ), GLUT-1 (tumour (b) , granuloma (e) ) and H E staining (tumour (c) , granuloma (f) ). White arrowhead : cancer cell; black arrow : lymphocyte infiltration; white arrow : epithelioid cell granuloma. Bars indicate 50 μm.

    Journal: EJNMMI Research

    Article Title: Differentiation of malignant tumours from granulomas by using dynamic [18F]-fluoro-L-α-methyltyrosine positron emission tomography

    doi: 10.1186/s13550-015-0109-z

    Figure Lengend Snippet: Microscopy images (×40) of immunofluorescent staining of LAT-1 (tumour (a) , granuloma (d) ), GLUT-1 (tumour (b) , granuloma (e) ) and H E staining (tumour (c) , granuloma (f) ). White arrowhead : cancer cell; black arrow : lymphocyte infiltration; white arrow : epithelioid cell granuloma. Bars indicate 50 μm.

    Article Snippet: LAT-1 was stained by using an anti-LAT-1 mAb (rabbit IgG, synthetic peptide, Abcam).

    Techniques: Microscopy, Staining

    Co-localization of GFP fluorescence and α-GFP immunofluorescence in tissue sections of cysticerci after 24 h of microinjection. a GFP fluorescence in a GFP-TOPO plasmid microinjected cyst (FITC filter); b Immunohistochemical localization using a 1:250 dilution of a polyclonal α -GFP rabbit IgG antibody followed by a 1:200 dilution of a goat α-rabbit IgG CY3-conjugated antibody (CY3 filter); c Merging of a and b images. d Water microinjected cysts (FITC filter); e Immunohistochemical localization as in b in a water microinjected cyst (CY3 filter); f Merging of d and e images. Arrows show subtegumentary cytons. T tegument. Photographs were obtained under Confocal Laser microscopy

    Journal: SpringerPlus

    Article Title: Transient transgenesis of the tapeworm Taenia crassiceps

    doi: 10.1186/s40064-015-1278-y

    Figure Lengend Snippet: Co-localization of GFP fluorescence and α-GFP immunofluorescence in tissue sections of cysticerci after 24 h of microinjection. a GFP fluorescence in a GFP-TOPO plasmid microinjected cyst (FITC filter); b Immunohistochemical localization using a 1:250 dilution of a polyclonal α -GFP rabbit IgG antibody followed by a 1:200 dilution of a goat α-rabbit IgG CY3-conjugated antibody (CY3 filter); c Merging of a and b images. d Water microinjected cysts (FITC filter); e Immunohistochemical localization as in b in a water microinjected cyst (CY3 filter); f Merging of d and e images. Arrows show subtegumentary cytons. T tegument. Photographs were obtained under Confocal Laser microscopy

    Article Snippet: Loading controls for western blots of T. crassiceps crude extracts were carried out using host albumin for reference (Aldridge et al. ); a sheep anti-mouse albumin polyclonal IgG antibody (Abcam) was used followed by a horseradish peroxidase conjugated anti-sheep IgG (Abcam) secondary antibody.

    Techniques: Fluorescence, Immunofluorescence, Plasmid Preparation, Immunohistochemistry, Microscopy

    Time course of the GFP fluorescence after microinjection of intact Taenia crassiceps cysticerci. a GFP-TOPO plasmid ( 1 – 3 ) and a GFP-negative plasmid: pCMV-VSV-G ( 4 ) microinjected cysts, after 24 ( 1 and 4 ); 48 ( 2 ) and 72 h ( 3 ). Photographs were taken using an Olympus DSU confocal system with a FITC (450–490 nm) filter under the same conditions for all cases. b Western blots using crude extracts of GFP-TOPO ( 1 – 3 ) and pCMV-VSV-G ( 5 ) microinjected cysts; 24 ( 1 and 5 ); 48 ( 2 ) and 72 ( 3 ) h post microinjection. A crude extract of HEK 293 cells transfected with GFP-TOPO ( 4 ) is also shown as a positive control. 50 µg of each crude extract were loaded on each lane in the gel and all blots were obtained from a single gel run. For detection of GFP, a polyclonal rabbit IgG α-GFP antibody and a goat α-rabbit IgG antibody conjugated with horseradish peroxidase were used at 1:1000 and 1:10,000 dilutions, respectively. Loading controls ( 6 – 10 ) used a sheep anti-mouse albumin polyclonal antibody followed by a horseradish peroxidase conjugated anti-sheep IgG secondary antibody (Aldridge et al. 2006 ). Development of peroxidase was carried out using an enhanced chemiluminescence kit

    Journal: SpringerPlus

    Article Title: Transient transgenesis of the tapeworm Taenia crassiceps

    doi: 10.1186/s40064-015-1278-y

    Figure Lengend Snippet: Time course of the GFP fluorescence after microinjection of intact Taenia crassiceps cysticerci. a GFP-TOPO plasmid ( 1 – 3 ) and a GFP-negative plasmid: pCMV-VSV-G ( 4 ) microinjected cysts, after 24 ( 1 and 4 ); 48 ( 2 ) and 72 h ( 3 ). Photographs were taken using an Olympus DSU confocal system with a FITC (450–490 nm) filter under the same conditions for all cases. b Western blots using crude extracts of GFP-TOPO ( 1 – 3 ) and pCMV-VSV-G ( 5 ) microinjected cysts; 24 ( 1 and 5 ); 48 ( 2 ) and 72 ( 3 ) h post microinjection. A crude extract of HEK 293 cells transfected with GFP-TOPO ( 4 ) is also shown as a positive control. 50 µg of each crude extract were loaded on each lane in the gel and all blots were obtained from a single gel run. For detection of GFP, a polyclonal rabbit IgG α-GFP antibody and a goat α-rabbit IgG antibody conjugated with horseradish peroxidase were used at 1:1000 and 1:10,000 dilutions, respectively. Loading controls ( 6 – 10 ) used a sheep anti-mouse albumin polyclonal antibody followed by a horseradish peroxidase conjugated anti-sheep IgG secondary antibody (Aldridge et al. 2006 ). Development of peroxidase was carried out using an enhanced chemiluminescence kit

    Article Snippet: Loading controls for western blots of T. crassiceps crude extracts were carried out using host albumin for reference (Aldridge et al. ); a sheep anti-mouse albumin polyclonal IgG antibody (Abcam) was used followed by a horseradish peroxidase conjugated anti-sheep IgG (Abcam) secondary antibody.

    Techniques: Fluorescence, Plasmid Preparation, Western Blot, Transfection, Positive Control