Journal: Frontiers in Physiology

Article Title: Increased Immunosignals of Collagen IV and Fibronectin Indicate Ischemic Consequences for the Neurovascular Matrix Adhesion Zone in Various Animal Models and Human Stroke Tissue

doi: 10.3389/fphys.2020.575598

Figure Lengend Snippet: Triple fluorescence staining: marker combinations concomitantly applied with Cy3-stained rabbit-anti-fibronectin*.

Article Snippet: guinea pig-anti-NeuN (1:200; Synaptic Systems; Göttingen, Germany) , Cy2-donkey-anti-guinea pig IgG , biotinylated STL (20 μg/ml; Vector) , Cy5-streptavidin.

Techniques: Fluorescence, Staining, Marker, Plasmid Preparation

Journal: Molecular cancer therapeutics

Article Title: Anti-KIT DNA Aptamer for Targeted Labeling of Gastrointestinal Stromal Tumor

doi: 10.1158/1535-7163.MCT-19-0959

Figure Lengend Snippet: (A-B) Immunocytochemistry and fluorescence microscopy of ex vivo GIST xenograft fragments. Representative images of anti-KIT or scrambled aptamer labeling of xenograft fragments. Fluorescence intensity quantification (Living Image, PerkinElmer) tumor fragments (N=6, p-value by Kruskal-Wallis test). (C) Schema demonstrating the experimental workflow of in vivo aptamer labeling in an intraperitoneal model of GIST, as well as representative images of GFP-labeled GIST-T1 xenograft in situ and Cy5.5-labeled aptamer. (D) Waterfall plot analysis of individual tumors with ratio of Cy5.5-labeled aptamer fluorescence intensity to GFP-labeled GIST-T1 xenograft fluorescence intensity (N=10). (E) Cy5.5-labeled aptamer fluorescence intensity (Living Image, PerkinElmer) normalized to tumor area for comparison of in vivo labeled IP model of GIST (p-value by Kruskal-Wallis test).

Article Snippet: The aptamer used for live imaging experiments were either directly conjugated to Cy5.5 or pre-conjugated with streptavidin-Cy5.5 (Rockland Immunochemicals, Limerick, PA) with a biotinylated aptamer.

Techniques: Immunocytochemistry, Fluorescence, Microscopy, Ex Vivo, Labeling, In Vivo, In Situ

Journal: Human Vaccines & Immunotherapeutics

Article Title: High proportions of regulatory B and T cells are associated with decreased cellular responses to pH1N1 influenza vaccine in HIV-infected children and youth (IMPAACT P1088)

doi: 10.4161/hv.23774

Figure Lengend Snippet: Figure 3. Kinetics of circulating CD4+CD25+ (B) and CD4+FOXP3+ (C) T cells after the two double-doses of pH1N1 vaccine. PBMC, frozen and thawed using procedures that preserve viability, were stained with mAbs anti-CD3, CD8, CD19, CD25 and FOXP3 and IL10 as described in the methods section. Panel A shows the gating strategy: (1) lymphocytes were identified by forward/side scatter; (2) CD4+ T cells were gated by expression of CD3 (CD3+) and lack of expression of CD8 (CD8-); (3) CD25+ and FOXP3+ CD4+ T cells were gated as shown. IL10 expression was gated using FOXP3 on one axis and IL10 on the other axis (not shown). CD8+ T cells were gated using expression of CD3 and CD8. B cells were gated as CD3-CD19+ (not shown). CD8+ T-cell and B-cell expression of CD25, FOXP3 and IL10 (not shown) were gated as described for CD4+ T cells. Panels B and C show only the lymphocyte subsets with significant changes over time. Results are presented as medians and IQRs. The number of subjects that contributed data at each time point are indicated on the graphs. Statistically significant differences assessed by Wilcoxon Matched Paired Signed Ranks test are shown on the graphs in panels B and C.

Article Snippet: Cells were stained using the following conjugated mAbs: anti-CD3- PECy7, anti- CD8-APC-AF 750, anti-CD19-PECy5, anti-IL-10-APC, anti-FOXP3-FITC, anti-TGFβ-PE (Cederlane; TB21), anti-MIP1β-PE (BD Biosciences; D21–1351), and anti-TNFα-PerCP Cy5.5 (Biolegend; MAb11), anti-Perforin-APC (Biolegend; dG9) and anti-IL2-FITC (BD Biosciences; 5344.111).

Techniques: Staining, Expressing

Journal: Human Vaccines & Immunotherapeutics

Article Title: High proportions of regulatory B and T cells are associated with decreased cellular responses to pH1N1 influenza vaccine in HIV-infected children and youth (IMPAACT P1088)

doi: 10.4161/hv.23774

Figure Lengend Snippet: Figure 4. Kinetics of pH1N1-stimulated CD8+FOXP3+ (A) and CD8+TGFβ+ (B) T cells after the two double-doses of pH1N1vaccine. PBMC with viability ≥ 70% were incubated for 48h with medium control and with live pH1N1 virus to promote stimulation through both MHC class I and class II. After incubation cells were stained with mAb anti-CD3, CD8, CD19, CD25, FOXP3 and IL10. Companion tubes were also stained with mAb anti-CD3, CD8, IL2, TNFα, MIP1β and TGFβ. The gating strategy (not shown) was similar to that described for freshly thawed, unstimulated PBMC (Fig. 3). The number of subjects that contributed data at each time point are indicated on the graphs. Results, depicted as medians and IQRs, are shown only for the lymphocyte populations with significant changes over time. Statistically significant differences assessed by Wilcoxon Matched Paired Signed Ranks test are shown on the graphs.

Article Snippet: Cells were stained using the following conjugated mAbs: anti-CD3- PECy7, anti- CD8-APC-AF 750, anti-CD19-PECy5, anti-IL-10-APC, anti-FOXP3-FITC, anti-TGFβ-PE (Cederlane; TB21), anti-MIP1β-PE (BD Biosciences; D21–1351), and anti-TNFα-PerCP Cy5.5 (Biolegend; MAb11), anti-Perforin-APC (Biolegend; dG9) and anti-IL2-FITC (BD Biosciences; 5344.111).

Techniques: Incubation, Staining

Journal: Human Vaccines & Immunotherapeutics

Article Title: High proportions of regulatory B and T cells are associated with decreased cellular responses to pH1N1 influenza vaccine in HIV-infected children and youth (IMPAACT P1088)

doi: 10.4161/hv.23774

Figure Lengend Snippet: Figure 6. Correlations of pH1N1 antibody titers and of IFNγ ELISPOT results with circulating CD19+CD25+% B cells, CD4+CD25+FOXP3+% and CD8+CD25+FOXP3+% Treg after the first immunization. Data were derived from 72, 66 and 66 participants in panels A­−C, respectively. The x axes represent the difference between baseline and post-dose 1 pH1N1 HAI (A) or IFNγ ELISPOT (B and C). HAI antibody titers to pH1N1 were measured as described in the methods section. PBMC, frozen and thawed to preserve viability, were rested overnight. ELISPOT assays were performed only on PBMC with viability ≥ 70% both immediately after thawing and after resting. The assays used MabTech kits and live pH1N1 viral infection to promote stimulation of both CD4+ and CD8+ T cells. For flow cytometric analysis of circulating B and T cells, freshly thawed PBMC with adequate viability were stained with mAbs anti-CD3, CD8, CD19, CD25 and FOXP3 and IL10 as described in the methods section. The graphs depict only the correlations that reached statistical significance using the Spearman correlation test. Correlation coefficients and p values are shown on the graph.

Article Snippet: Cells were stained using the following conjugated mAbs: anti-CD3- PECy7, anti- CD8-APC-AF 750, anti-CD19-PECy5, anti-IL-10-APC, anti-FOXP3-FITC, anti-TGFβ-PE (Cederlane; TB21), anti-MIP1β-PE (BD Biosciences; D21–1351), and anti-TNFα-PerCP Cy5.5 (Biolegend; MAb11), anti-Perforin-APC (Biolegend; dG9) and anti-IL2-FITC (BD Biosciences; 5344.111).

Techniques: Enzyme-linked Immunospot, Derivative Assay, Infection, Staining