Cd4 monoclonal antibody

Immunohistochemistry stainings were performed on paraffin-embedded spleens and mLNs from mice. Following deparaffinization, antigen retrieval and blocking, the slides were incubated with the primary antibody diluted 1:200 in TBST with 2% BSA overnight at 4 °C (CD4 Monoclonal Antibody (4SM95), eBioscience™ (Invitrogen) or Recombinant Anti-CD8 alpha antibody (Abcam)) and with the secondary antibody diluted 1:1000 in TBS for 1 h at room temperature (AffiniPure Goat Anti-Rabbit IgG (H + L) (Jackson ImmunoResearch) or Biotin Goat Anti-Rat Ig Clone Polyclonal (RUO) (554014) (BD Biosciences)). A negative control was included by not adding first antibody on one tissue sample from each slide. Non-overlapping pictures (≥ 5 pictures per mLN (n ≥ 2) and ≥ 7 pictures per spleen (n = 3) per staining (CD4+ or CD8+)) of the whole organs were taken with the Leica DMI4000 B inverted microscope and analysed with ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/, 1997–2018.). There, the two color channels were separately thresholded to generate a binary image, and the area fraction of the DAPI staining and the positive staining (CD4+ or CD8a+) were measured. Subsequently, the ratio between the CD4+ or CD8a + area relative to the area of DAPI staining was calculated.

Cells in single-cell suspension were fixed with 2% PFA (Santa-Cruz, #sc-281692). After washing with a Flow Cytometry Staining Buffer (eBioscience, #00–4222–26), cells were stained with antibodies for cell-surface marker for 1 h on ice in the dark. For staining of intracellular proteins, the cells were washed and resuspended in the permeabilization buffer (BD, #554723) and stained by antibodies in the permeabilization buffer for 1 h on ice in the dark. The cells were then pelleted and resuspended in the Flow Cytometry Staining Buffer for flow cytometry analysis. Antibodies used for flow cytometry include: V450 Mouse Anti-Mouse CD45.2 (BD Horizon, #560697, Clone 104), APC anti-mouse NK1.1 Antibody (BioLegend, #108710, clone PK136), FITC anti-human/mouse Granzyme B Antibody (BioLegend, #515403, clone GB11), CD8a Monoclonal Antibody (53–6.7) PE-Cyanine7 (eBioscience, #25–0081–82, clone 53–6.7), CD4 Monoclonal Antibody (RM4–5), and PE (eBioscience, #12–0042–82, clone RM4–5).