Cd68 clone kp 1

Immunohistochemistry (IHC) was performed on serial cuts of the FFPE biopsies (3–4 µm). For viral antigens, LMP1 (CS1-4 pool of clones, Dako) and EBNA2 (1E6 y R3 clones, Abcam) primary antibodies as latency antigens and the BMRF1 primary antibody (G3-E31 clone, Abcam) as an early lytic antigen were used as described (16 (link)). Cytokine detection was assessed with specific primary antibodies against IFN-γ (polyclonal, Abcam), IL-10 (Ab34843, Abcam), and TGF-β (Ab9758, Abcam) as described (8 (link)). Lastly, a double-staining for macrophage polarization markers CD68 (clone KP-1, Roche Ventana), CD163 (clone MRQ-26, Roche Ventana), and PD-L1 (clone 4E54, Abcam) was accomplished. Antigenic retrieval was performed using sodium citrate buffer pH = 6.0. After incubation with the first primary antibody (CD68 or CD163), immunodetection was performed using a commercial kit (ScyTek) followed by diaminobenzidine (DAB) as a chromogen. Then, the second primary antibody PD-L1 diluted in 1/100 TBS-BSA 2% was incubated overnight, and antibody signal amplification was performed using Alexa 594 anti-mouse, followed by Hoescht.

Antibodies against TIM3 (polyclonal, Abcam), LAG3 (clone [11E3], Abcam), CD68 (clone KP-1, Roche Ventana) and CD163 (clone MRQ-26, Roche Ventana) were used, as described [10 (link), 14 (link)], to characterize microenvironment composition and to validate gene expression analysis. IHC detection of antibodies was carried out using a universal streptavidin–biotin complex-peroxidase detection system (UltraTek HRP Anti-Polyvalent Lab Pack, ScyTek, UT) according to the manufacturer’s instructions. Visualization of positive cells was performed using diaminobenzidine as chromogen. Appropriate positive controls were immunostained for each antibody. The counting of TIM3, LAG3, CD68 and CD163 positive cells was performed as follows: using the 100 × objective lenses and counting ten fields selected on the basis of the best-preserved tissue areas that contained immunopositive cells. The positive cells in tumor cells and in tumor-infiltrating lymphocytes (TILs) were counted, and the results were expressed as positive cells per area unit (cells + /mm2). Cells partly included in the fields were not counted.

We performed immunohistochemical stains on formalin-fixed paraffin-embedded three micron tissue sections using an automated immunostainer Benchmark Ultra (Roche) with an Ultra-View Universal DAB brown detection kit (alongside an Ultra-View Universal AP Red detection kit for double staining). These antibodies were used for the following targets: CD3 (clone 2GV6, predilute Roche Cat # 790-4341), CD4 (clone SP35, predilute Roche Cat# 790-4423), CD8 (clone SP57, predilute Roche Cat#790-4460), granzyme B (clone GrB-7+D170, dilution 1:100, Millipore Cat# MAB 3070), perforin (clone KM585 PI-8) (dilution 1:200, Leica Cat# NCL-L-perforin) detection with Optiview kit (Roche), CD68 (clone KP1, predilute Roche Cat#790-2931), CD163 (clone MRQ-26, predilute Roche Cat#760-4437).