IHC analysis
SECONDARY
Serial 3 µm formalin-fixed paraffin-embedded tissue sections were stained using an AutostainerPlus (Dako). Antigen retrieval and deparaffinization were carried out on a PT-Link (Dako) using the EnVision FLEX Target Retrieval Solutions (Dako). Endogenous peroxidase and non-specific staining were blocked with H202 3% (Gifrer, 10603051) and Protein Block (Dako, X0909) respectively. The antibodies used are: anti-CD8 clone C8/144B (M7103, Dako) at a final concentration of 3.14 µg/mL and anti-HLA Class I, clone EMR8-5 (ab70328, abcam) at a final concentration of 0.3 µg/mL. The HRP-labeled polymer conjugated EnVision+ Single Reagent (Dako, K4001) was used as a secondary antibody. Peroxidase activity was detected using 3-amino-9-ethylcarbazole substrate (Vector Laboratories, SK-4200). All stained slides were digitalized with a NanoZoomer scanner (Hamamatsu).