IHC
dehydration, tumor
DEHYDRATION, SECONDARY, TUMOR
We performed immunohistochemical analyses for myeloid cell markers (Iba-1, CD68 and CD163) and lymphoid cell markers (CD3, CD4 and CD8) in samples with available tissue before and after treatment in this sample. Staining and subsequent annotation and analyses were performed blinded to clinical status. Slides with 5 µm FFPE tissue sections were rehydrated and a sodium citrate-dihydrate buffer or Tris–EDTA buffer was used for heat-mediated antigen retrieval. A 3% hydrogen peroxide in methanol solution was utilized to block endogenous peroxidase activity. Blocking solution (5% bovine serum albumin in phosphate buffered saline plus 0.1% Triton X-100) was applied to slides for 1 h at room temperature. Subsequently, primary antibodies including anti-CD3 (Agilent, M725401-2, mouse monoclonal, 1:100), anti-IBA1 (Wako, 019-19741, rabbit polyclonal, 1:1,500), anti-CD68 (Agilent, M0514, mouse monoclonal, 1:200), anti-CD4 (abcam, ab133616, rabbit monoclonal, 1:100) and anti-CD8 (abcam, ab93278, rabbit monoclonal, 1:250) were applied overnight at 4 °C in blocking solution. A 1 h incubation with secondary antibody was performed followed by processing with the DAKO polymer-HRP system and DAB peroxidase kit, counterstaining with hematoxylin, dehydration of the tissue and coverslipping. Whole slide images were digitized, and then for each slide tumor versus non tumor content was annotated and representative images were selected. Proportions of stain-positive cells were quantified using HALO (version 3.0311, Indica Labs) software algorithms that were defined to identify cells with either nuclear or cytoplasmic staining as a fraction of all cells. This algorithm was applied to all annotated tissue sections in an unbiased systematic manner, and the density of immunopositivity per square millimeter was recorded for each antibody. PD-L1 protein expression was performed by NeoGenomics Laboratories (NeoGenomics) under the direction of Merck using FFPE tumor biopsy samples according to standard protocols (PD-L1 IHC 22C3 assay).