Anti cd8 sp57

An automatic stainer (BenchMark ULTRA, Ventana Medical System, Inc.) was used for the immunohistochemical test. The antigen was retrieved with cell conditioning buffer 1. Next, endogenous peroxidase was inhibited with H₂O₂ at 3% (Bioptica) for 10 min. Samples were incubated with primary antibody anti-CD3 (2GV6) (Roche-Ventana), anti-CD8 (SP57) (Roche-Ventana), Rabbit Monoclonal Pre-diluted (0.4μg/mL), for 20 min at 36°C; anti-CD20 (L26) (Roche-Ventana), Mouse Monoclonal Pre-Diluted (0.4μg/mL) for 24 min at 36°C. The antibody was exposed with ultraView Universal DAB Detection Kit (Cat No. 760–500). As counterstain, Mayer haematoxylin was used for 4 min.
TIL levels were assessed by two investigators blind to the patients’ clinical-pathological data using the standardized method coded in 2015 by the International TILs Working Group [13 (link)]. TILs were investigated per microscopic field (5X and 10X) and an average over ten independent regions having the most abundant immunoreactive cells was calculated for each slide.

Immunohistochemistry (IHC) was performed using histological tissue microarray slides that were 3 µm thick. The following antibodies were used: anti-PD-L1 (clone CAL10, Master Diagnostica, Granada, Spain) and anti-CD8 (SP57, Ventana, AZ, USA). The sections were stained using the Ventana BenchMark Ultra Autostainer (Ventana, Tucson, AZ-85755, USA) and the UltraView Universal DAB kit (Ventana, Tucson, AZ-85755, USA), following the manufacturer’s instructions. PD-L1 expression in cancer cells (CCs) and TILs, as well as the density of CTLs, were evaluated by IHC. When evaluating PD-L1 expression, non-tumoral colorectal mucosa were utilized as internal negative controls. Tonsillectomy materials in the pathology archive were used as positive controls.