Anti human cd8 c8 144b

Five μm-thick formalin-fixed paraffin-embedded (FFPE) tissue sections on glass slides were backed at 37°C overnight, deparaffinized in xylene, and rehydrated in decreasing concentrations of ethanol. Then, tissue sections were incubated in retrieval solution (Citrate buffer, pH6) for antigen retrieval at 95°C for 30 minutes. Tissue sections were incubated in 3% hydrogen peroxide and in serum-free protein block solution (Dako, X0909) before adding the primary antibody (CD68, clone KP1, Dako) for 1 hour at room temperature. After signal amplification using biotinylated-secondary antibody and streptavidin-horseradish peroxidase, chromogenic revelation was performed using 3-amino-9-ethylcarbazole (AEC). Slides were counterstained with hematoxylin, mounted with a glycerol-based mounting medium and scanned for digital imaging (Pannoramic 250 Flash III whole-slide scanner, 3DHISTECH). Then the same slides were successively stained as described (Remark et al., 2016 (link)). Primary antibodies were: anti-human CD3 (2GV6, Ventana), anti-human CD8 (C8/144b, Dako), anti-human CD20 (L26, Dako), anti-human DC-LAMP (1010E1.01, Novus biologicals), anti-human PD-L1 (E1L3N, Cell Signaling Technologies), anti-human CD206 (polyclonal, Abcam), and anti-human HLA Class I (EMR8-5, Abcam).