Comprehensive Immunohistochemical Profiling of Tumor Microenvironment

Immunohistochemistry (IHC) analysis was used to detect the MMR-related proteins MSH2, MSH6, MLH1, and PMS2. To assess the TIME, CD3, CD8, and PD-L1 expression were evaluated. IHC was performed using our laboratory protocol as described previously [22 (link), 23 (link)]. Briefly, 3-μm-thick TMA serial sections were deparaffinized and subjected to heat-induced epitope retrieval with 10 mM sodium citrate (pH 6.0) at 95°C for 20 min. Endogenous peroxidase activity was quenched using a 0.3% hydrogen peroxide solution.
TMA sections were incubated with primary antibodies against MLH1 (clone ES05, ready to use; Leica Biosystems), PMS2 (clone MOR4G, ready to use; Leica Biosystems), MSH2 (clone 25D12, ready to use; Leica Biosystems), MSH6 (clone PU29, ready to use; Leica Biosystems), CD3 (clone LN10, ready to use; Leica Biosystems), CD8 (clone 4B11, ready to use; Leica Biosystems), and PD-L1 (SP142, 1: 100, ZSGB-BIO, China). Human tonsils treated with primary antibodies were used as positive controls, while the same tissues without primary antibodies comprised the negative controls. After the reactions, all sections were counterstained with hematoxylin. All slides except those used for manual PD-L1 staining were stained using an automatic IHC staining instrument (BOND-III; Leica Biosystems, Wetzlar, Germany) according to the manufacturer's instructions.