Immunohistochemical Analysis of Tissue Microarrays

IHC analyses of tissue microarrays were performed, as described previously [16 (link)]. Briefly, samples were deparaffinized in xylene and dehydrated in a graded series of ethanol. After rinsing in TBS buffer, the samples were processed for antigen retrieval in a 1 mM EDTA retrieval solution (pH 9.0) (Nichirei Co.) at 95 °C for 40 min. Endogenous peroxidase was then blocked by incubation in 0.3% H₂O₂-methanol at room temperature for 10 min. After rinsing with TBS, samples were incubated with primary antibodies against human PD-L1 (1:200) and p-FAK (1:100) for 30 min, and then with EnVision (Dako Corp) for 30 min. After rinsing in TBS, the coloring reaction was performed with DAB (Dojindo) for 5 min. Each sample was counterstained with hematoxylin. These processes were all performed at room temperature. For sirius red staining, tissue slides were dewaxed, dehydrated, and stained with Picro-sirius red for 1 h (Sigma-Aldrich, S365548). After staining, slides were washed in two dips of acidified water and dehydrated with 100% ethanol. Finally, slides were cleared in xylene and mounted. Quantification of collagen staining was performed using Image J software [7 (link)].