Journal: Journal of Neuroinflammation

Article Title: Reactive microglia and IL1β/IL-1R1-signaling mediate neuroprotection in excitotoxin-damaged mouse retina

doi: 10.1186/s12974-019-1505-5

Figure Lengend Snippet: Antibodies, sources, and working dilutions

Article Snippet: Ki67 , 1:300 , Rabbit , DRM004 , OriGene.

Techniques: Plasmid Preparation

Journal: Journal of Neuroinflammation

Article Title: Reactive microglia and IL1β/IL-1R1-signaling mediate neuroprotection in excitotoxin-damaged mouse retina

doi: 10.1186/s12974-019-1505-5

Figure Lengend Snippet: IL1β stimulates the reactivity and proliferation of microglia in the retina and conveys neuroprotection following NMDA-treatment. Eyes were injected with saline or IL1β, and retinas harvested 24 h later ( a – e ). Eyes were injected with saline or IL1β on day 1, NMDA on day 2, and retinas harvested 1 or 12 days later ( f - m ). Retinal sections were labeled with TUNEL ( f ) or DRAQ5 (red; k ) or antibodies to F4/80 (green) and Iba1 (red; a ), tomato lectin (green; d ) and Ki67 (red; d ), calretinin (green; k ), or F4/80 (green) and Iba1 (red; l ). The box plots in b , c , e, g–j, and m illustrate the mean, upper extreme, lower extreme, upper quartile, and lower quartile ( n ≥ 6 animals). Significance of difference (* p < 0.05) was determined by using a t test. Arrows indicate microglia. The calibration bars in panels a, d, f, k, and l represents 50 μm. Abbreviations: ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer

Article Snippet: Ki67 , 1:300 , Rabbit , DRM004 , OriGene.

Techniques: Injection, Saline, Labeling, TUNEL Assay

Journal: Scientific Reports

Article Title: Breast Cancer Cell Invasion into a Three Dimensional Tumor-Stroma Microenvironment

doi: 10.1038/srep34094

Figure Lengend Snippet: ( A ) Cells were stained for EGFR (red), pEGFR (green), and nuclei (blue) (scale bar: 20 μm). ( B ) Representative images of EGFR clusters with corresponding heat maps of relative intensities. ( C ) (+) EGF demonstrated significantly lower EGFR to cell area ratio. ( D , E ) (+) EGF displayed significantly higher pEGFR to cell area ratio and pEGFR to EGFR area ratio ( p < 0.05 calculated from Student’s t -test with n > 18 from more than three devices for each condition).

Article Snippet: Next, IF buffer (0.2% (v/v) Triton X-100 + 0.1% (v/v) BSA (radioimmunoassay grade) + 0.05% Tween 20, 7.7 mM NaN 3 in PBS) + 10% (v/v) goat serum was added into the channels and the devices were incubated at room temperature for 1.5 h. Later, primary antibodies, monoclonal Anti-α-Tubulin (1:500, T9026, Sigma-Aldrich), Ki-67 (1:100, (DSHB Hybridoma Product AFFN-KI67-3E6)), EGFR (1:1000, MA5-13319, Thermo Scientific), or pEGFR (1:100, 3777S, Cell Signaling Technology ® ) were diluted at in IF buffer and devices were parafilmed and kept at 4 °C overnight.

Techniques: Staining

Journal: Orphanet Journal of Rare Diseases

Article Title: Activation of PKC triggers rescue of NPC1 patient specific iPSC derived glial cells from gliosis

doi: 10.1186/s13023-017-0697-y

Figure Lengend Snippet: Analysis of gliosis marker. a - d NPC1 mutant cell lines contained a higher amount of GFAP + and vimentin + cells (red, a - d ). DAPI staining (blue) indicates nuclei. Scale 100 μm. ( e ). Colocalization analysis of GFAP and vimentin revealed a significantly increased amount of double positive cells in all NPC1 mutant cell lines. f FACS analysis of GFAP + /vimentin + cells confirmed an increased amount of glia cells in NPC1 mutant cell lines ( N = 5–7, n = 14–23). g In addition, semi-quantitative protein measurement by western blot demonstrated a higher amount of GFAP ( N = 7–11, n = 26–43) and h an increased amount of vimentin in NPC1 mutant cells ( N = 5–7, n = 14–23). i FACS analysis of cells positive for GFAP and Ki67, elucidated an increased amount of double positive cells indicating gliosis NPC1 mutant cell lines ( g ; N = 3–4, n = 7–9)

Article Snippet: For analysis cells were incubated with primary antibodies, GFAP (1:500, rabbit IgG, DAKO, Germany or 1:100, Rat IgG, Thermo Scientific, USA), Vimentin (1:100, mouse IgG, Invitrogen, Germany), Ki67 (1:100, rabbit IgG, Santa Cruz Biotechnology, Germany) in Saponin buffer (0.5% Saponin, 0,5% BSA, 0.02% NaN3) for 2 h gently shaking.

Techniques: Marker, Mutagenesis, Staining, Western Blot

Journal: Orphanet Journal of Rare Diseases

Article Title: Activation of PKC triggers rescue of NPC1 patient specific iPSC derived glial cells from gliosis

doi: 10.1186/s13023-017-0697-y

Figure Lengend Snippet: Induction of gliosis by U18666A. a Unaffected control cells were treated with ( b ) U18666A to induce cholesterol accumulations shown by Filipin staining (blue). Scale 100 μm. c Calculation of relative fluorescence confirmed higher cholesterol amount in U18666A treated cells ( N = 4, n = 6–8). d Increased cholesterol amount in U18666A treated cells was also detected by means of the Amplex red assay ( N = 4, n = 7–8). e FACS analysis of GFAP + /vimentin + cells and f GFAP + /Ki67 + cells revealed an increased amount of reactive astrocytes ( N = 4, n = 7–12). g Treatment with U18666A resulted in a reduced amount of p-vimentin and ( h ) p-GFAP ( N = 4, n = 8–12)

Article Snippet: For analysis cells were incubated with primary antibodies, GFAP (1:500, rabbit IgG, DAKO, Germany or 1:100, Rat IgG, Thermo Scientific, USA), Vimentin (1:100, mouse IgG, Invitrogen, Germany), Ki67 (1:100, rabbit IgG, Santa Cruz Biotechnology, Germany) in Saponin buffer (0.5% Saponin, 0,5% BSA, 0.02% NaN3) for 2 h gently shaking.

Techniques: Control, Staining, Fluorescence, Amplex Red Assay

Journal: Orphanet Journal of Rare Diseases

Article Title: Activation of PKC triggers rescue of NPC1 patient specific iPSC derived glial cells from gliosis

doi: 10.1186/s13023-017-0697-y

Figure Lengend Snippet: Effect of PMA on gliosis. a Cells were treated with 10 nM PMA and number of GFAP + /vimentin + cells and ( b ) GFAP + /Ki67 + cells was quantified. FACS analysis revealed a significant reduction of GFAP + /vimentin + cells in all NPC1 mutant cell lines after PMA treatment ( N = 4–5, n = 11–28). Number of GFAP + /Ki67 + cells ( b ) was reduced in all NPC1 mutant cell lines after treatment with PMA ( N = 4–5, n = 7–16). Asterisks above bars indicate significance to untreated control and asterisks above lines indicate significances between treated and untreated cells

Article Snippet: For analysis cells were incubated with primary antibodies, GFAP (1:500, rabbit IgG, DAKO, Germany or 1:100, Rat IgG, Thermo Scientific, USA), Vimentin (1:100, mouse IgG, Invitrogen, Germany), Ki67 (1:100, rabbit IgG, Santa Cruz Biotechnology, Germany) in Saponin buffer (0.5% Saponin, 0,5% BSA, 0.02% NaN3) for 2 h gently shaking.

Techniques: Mutagenesis, Control

Journal: BioTechniques

Article Title: Simulated microgravity culture system for a 3-D carcinoma tissue model.

doi: 10.2144/02335rr02

Figure Lengend Snippet: Figure 3. Apoptosis and NF-κB activation in cells. (A) TUNEL assay. Representative pictures (400×; arrows, TUNEL-positive cells). (B) Results are expressed as the x– ± SD of three independent experiments. (C) Immunohistochemistry for NF-κB. Representative pictures (400×; arrows, nuclear translocation of NF-κB/p65).

Article Snippet: The slides were washed in PBS, and endogenous peroxidases were quenched in 3% H2O2 methanol for 30 min. Primary antibodies, anti-Ki-67 (1:50; Dako, Glostrup, Denmark) and anti-NF-κB p65 (1:50; Santa Cruz Biotechnology, CA, USA), were diluted with PBS supplemented with 0.01% NaN3 and 1% BSA and applied to sections, which were incubated overnight at 4°C.

Techniques: Activation Assay, TUNEL Assay, Immunohistochemistry, Translocation Assay

Journal: Frontiers in Oncology

Article Title: Gastric-type endocervical adenocarcinoma: a case report and literature review

doi: 10.3389/fonc.2024.1341068

Figure Lengend Snippet: Histopathological findings in the patient of G-EAC. (A) H&E staining demonstrates the histological features of G-EAC (400X). The tumor cells exhibit irregular glandular structures, invasive growth pattern, and cytoplasmic features characterized by transparency and a foamy appearance. The nuclei display irregular distribution along the basal portion of the glands. (B–D) IHC staining of the primary tumor cells showed that Ki-67, MUC6 and PAX8 were positive expression (400X). (E) P53 mutations were observed in the case.

Article Snippet: Simultaneously, immunohistochemistry (IHC) was used to analyze the expression of Ki-67 (1:150 dilutions; #ZM-0167, Zhong Shan Golden Bridge Biological Technology Inc.), Mucin 6 (MUC6) (1:200 dilutions; #ZM-0396, Zhong Shan Golden Bridge Biological Technology Inc.), Paired Box 8 (PAX8) (1:150 dilutions; #ZM-0468, Zhong Shan Golden Bridge Biological Technology Inc.) and p53 (1:150 dilutions; #ZM-0408, Zhong Shan Golden Bridge Biological Technology Inc.).

Techniques: Staining, Immunohistochemistry, Expressing

Journal: Yonago Acta Medica

Article Title: Cerebellar Ganglioglioma in Childhood: Histopathologic Implications for Management During Long-term Survival: A Case Report

doi: 10.24563/yam.2017.12.008

Figure Lengend Snippet: Histopathologic finding of the cerebellar tumor surgically resected at 6 years of age. A and B: Hematoxylin–Eosin (HE) staining; and immunostaining of C: synaptophysin, D: glial fibrillary acidic protein, E and F: phosphorylated S6, G: phosphatase and tensin homologue deleted on chromosome 10 (PTEN); counterstained with HE, H: phosphorylated PTEN. A, F, I–K, Bar = 50 µm; B–D, G, Bar = 25 µm; E and H, Bar = 100 µm.

Article Snippet: The sections were incubated with the primary antibody against synaptophysin (mouse monoclonal, diluted at 1:100; Boehringer-Mannheim, Indianapolis, IN), glial fibrillary acidic protein (mouse monoclonal, ready-to-use; BioGenex, San Ramon, CA), phosphorylated S6 ribosomal protein (P-S6) (rabbit polyclonal, diluted at 1:200; Cell Signaling Technology, Danvers, MA), PTEN (mouse monoclonal, diluted at 1:100; Santa Cruz, Dallas, TX), phosphorylated-PTEN (rabbit monoclonal, OriGene, Rockville, MD), Ki-67 (mouse monoclonal, ready to use, Clone MIB-1, DAKO, CA), or O6-methulguanine-DNA-methyltransferase (MGMT) (mouse monoclonal, ready to use, abcam, Cambridge, UK) overnight at 4 oC.

Techniques: Staining, Immunostaining

Journal: Cancers

Article Title: Adipose Stromal Cell-Derived Cancer-Associated Fibroblasts Promote Pancreatic Adenocarcinoma Progression Through SFRP4 Signaling

doi: 10.3390/cancers18020233

Figure Lengend Snippet: PDAC cell-induced ASC-CAF transition. ( A ), ASCs expressing RFP cultured with or without Capan-1 cells expressing GFP co-seeded at a 1:1 ratio. ( B ), Changes in RFP+ fibroblasts induced by Capan-1 co-culture after 7 days identified by IPA focused on SHH (right) and Wnt (left) signaling pathways. Changes in ASC/Capan-1 co-culture vs. ASCs alone (legend) indicate SHH and Wnt target activation. ( C ), The heatmap shows gene-wise scaled expression values of two potential therapeutic targets and aCAF genes in two biological replicates of ASCs with/without Capan-1 cell co-culture. Scale bar: gene-wise z-score of relative gene expression from high (red) to low (blue). ( D ), IF analysis of ASC/Capan-1 co-culture demonstrating COL11A1 expression in CDH1-negative fibroblasts (red, arrowheads). CDH1+ (green): cancer cells. Blue: nuclei. Scale bar: 100 μM. ( E ), RT-PCR analysis of mRNA expression in RFP+ cells sorted from ASCs with/without Capan-1 co-culture demonstrating the induction of LINC01614 and SFRP4 expression. Plotted: mean +/− SD, * p < 0.05, Student’s t -test.

Article Snippet: Upon blocking, the following primary antibodies were used (4 °C, 12 h): E-cadherin (CDH1) antibody 610181 (BD Biosciences) at 1:100; Ki-67 antibody 14-5698-80 (Invitrogen; Carlsbad, CA, USA) at 1:500; SFRP4 antibody HPA009712 (Sigma) at 1:100; COL11A1 antibody orb767627 (Biorbyt; Wuhan, Hubei, China) at 1:100; and -GFP (Gene Tex, #GTX26673; Irvine, CA, USA) at 1:250.

Techniques: Expressing, Cell Culture, Co-Culture Assay, Protein-Protein interactions, Activation Assay, Biomarker Discovery, Gene Expression, Reverse Transcription Polymerase Chain Reaction

Journal: Cancers

Article Title: Adipose Stromal Cell-Derived Cancer-Associated Fibroblasts Promote Pancreatic Adenocarcinoma Progression Through SFRP4 Signaling

doi: 10.3390/cancers18020233

Figure Lengend Snippet: The effect of LINC01614 ko and SFRP4 ko in ASC-CAFs. ( A ), RT-PCR analysis of mRNA expression in parental, LINC01614 -ko, and SFRP4 -ko ASCs confirming reduction in the respective target gene expression. ( B ), RT-PCR analysis of mRNA expression in parental, LINC01614 -ko, and SFRP4 -ko RFP+ ASCs sorted from co-culture with GFP+ Capan-1 cells, confirming reduction in the respective target gene expression. A-B, Plotted: mean +/− SD, * p < 0.05, ** p < 0.01, *** p < 0.005, ANOVA. ( C ), Top 15 GO terms identified from the comparison between parental ASCs and LINC01614 -ko ASCs after Capan-1 co-culture. DEGs were filtered based on adjusted p -values (<0.05) and ranked by log 2 fold change, with the top 100 DEGs selected for analysis. ( D ), Knockout of LINC01614 or SFRP4 inhibits the expression of aCAF genes ( C) induced in Capan-1 co-culture, as measured by RNAseq data. The heatmap shows gene expression values averaged across samples within each condition. Expression values are scaled by gene-wise to highlight relative differences between conditions. Scale bar: gene-wise z-score of relative gene expression from high (red) to low (blue). ( E ), IF analysis of ASCs co-cultured with Capan-1 cells (arrows), revealing COL11A1 expression (red arrowheads) reduced (pink arrowheads) upon LINC01614 ko and SFRP4 ko. Blue: nuclei. Scale bar: 100 μM. White arrowheads: Capan-1 cell colonies.

Article Snippet: Upon blocking, the following primary antibodies were used (4 °C, 12 h): E-cadherin (CDH1) antibody 610181 (BD Biosciences) at 1:100; Ki-67 antibody 14-5698-80 (Invitrogen; Carlsbad, CA, USA) at 1:500; SFRP4 antibody HPA009712 (Sigma) at 1:100; COL11A1 antibody orb767627 (Biorbyt; Wuhan, Hubei, China) at 1:100; and -GFP (Gene Tex, #GTX26673; Irvine, CA, USA) at 1:250.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Targeted Gene Expression, Co-Culture Assay, Comparison, Knock-Out, Gene Expression, Cell Culture

Journal: Cancers

Article Title: Adipose Stromal Cell-Derived Cancer-Associated Fibroblasts Promote Pancreatic Adenocarcinoma Progression Through SFRP4 Signaling

doi: 10.3390/cancers18020233

Figure Lengend Snippet: The effect of SFRP4 ko on SMAD-dependent PDAC progression in mice. ( A ), IF on adherent ASCs demonstrating that SFRP4 expression (red arrows) is missing in cells from SFRP4 -ko mice. ( B ), IF on KPC tumor sections demonstrating that SFRP4 expression (red arrows) is missing in CAFs of SFRP4 -ko mice. CDH1+ (green): cancer cells. ( C ), Weights (mean +/− SD) and pictures of KPC tumors resected from WT and SFRP4 -ko mice after 3 weeks. * p < 0.05, Student’s t -test. ( D ), Changes in body weight (mean +/− SD) from grafting day in WT and SFRP4 -ko mice after 3 weeks. ( E ), IF on KPC tumor sections showing that the frequency of proliferating Ki-67+ cells (red) is lower in tumors of SFRP4 -ko mice. ( F ), Quantification of metastatic KPC cells based on the numbers of colonies formed by adherent cells from liver cell suspensions. Graphs: mean colony number/well +/− SD. ( G ), Images of representative wells used for ( F ). * p < 0.05, Student’s t -test. ( H ), Representative sections of KPC tumors stained with Trichrome to reveal collagen deposition (blue arrows). N: necrosis; arrowheads: glandular epithelial structures increased in tumors of SFRP4 -ko mice. ( I ), Representative sections of KPC tumors subjected to COL11A1 and CDH1 IF. Green arrows: epithelial CDH1 expression. Red arrows: stromal COL11A1 expression reduced in SFRP4 -ko mice. Blue: nuclei. Scale bar: 100 μM.

Article Snippet: Upon blocking, the following primary antibodies were used (4 °C, 12 h): E-cadherin (CDH1) antibody 610181 (BD Biosciences) at 1:100; Ki-67 antibody 14-5698-80 (Invitrogen; Carlsbad, CA, USA) at 1:500; SFRP4 antibody HPA009712 (Sigma) at 1:100; COL11A1 antibody orb767627 (Biorbyt; Wuhan, Hubei, China) at 1:100; and -GFP (Gene Tex, #GTX26673; Irvine, CA, USA) at 1:250.

Techniques: Expressing, Staining

Journal: Cancers

Article Title: Adipose Stromal Cell-Derived Cancer-Associated Fibroblasts Promote Pancreatic Adenocarcinoma Progression Through SFRP4 Signaling

doi: 10.3390/cancers18020233

Figure Lengend Snippet: SMAD4 ko overrides the SFRP4 dependency of PDAC progression in mice. ( A ), IF on SMAD4 -ko KPC tumor sections demonstrating that SFRP4 expression (red arrows) is missing in CAFs of SFRP4 -ko mice. CDH1+ (green): cancer cells. ( B ), Weights (mean +/− SD) and pictures of SMAD4 -ko KPC tumors resected from WT and SFRP4 -ko mice after 3 weeks. ( C ), Changes in body weight (mean +/− SD) from grafting day in WT and SFRP4 -ko mice after 3 weeks. ( D ), Quantification of metastatic SMAD4 -ko KPC cells based on numbers of colonies formed by adherent cells from liver cell suspensions. Graphs: mean colony number/well +/− SD. ( E ), Images of representative wells used for ( D ). ( F ), Representative sections of SMAD4 -ko KPC tumors stained with Trichrome to reveal collagen deposition (blue arrows). ( G ), Representative sections of SMAD4 -ko KPC tumors subjected to COL11A1 and CDH1 IF. Red arrows: stromal COL11A1 expression. Green arrows: epithelial CDH1 expression. Blue: nuclei. Scale bar: 100 μM.

Article Snippet: Upon blocking, the following primary antibodies were used (4 °C, 12 h): E-cadherin (CDH1) antibody 610181 (BD Biosciences) at 1:100; Ki-67 antibody 14-5698-80 (Invitrogen; Carlsbad, CA, USA) at 1:500; SFRP4 antibody HPA009712 (Sigma) at 1:100; COL11A1 antibody orb767627 (Biorbyt; Wuhan, Hubei, China) at 1:100; and -GFP (Gene Tex, #GTX26673; Irvine, CA, USA) at 1:250.

Techniques: Expressing, Staining