Simple stain max po kit

The tumor PD-L1 and major histocompatibility (MHC)-class I expression, and CD8 þ T cell, CD20 þ B cell, and CD163 þ M2-macrophage infiltration in tumor tissues before anti-PD-1 therapy were analyzed by immunohistochemistry (IHC). 22 Details of analysis methods are fully provided in the Supplementary Materials. Tumor NY-ESO-1 and XAGE1 antigen expression before anti-PD-1 therapy were also analyzed by IHC. Four-micrometer-thick sections were deparaffinized with xylene and ethanol. Antigen retrieval was performed by microwave heating in antigen retrieval buffer (10 mM citrate buffer, pH 6.0) with a pressure cooker for 10 minutes. After the inactivation of endogenous peroxidase with 0.3% H 2 O 2 for 15 and 5 minutes, respectively, specimens were pre-incubated with serum-free blocking solution (Nacalai Tesque and DakoCytomation, Kyoto, Japan) for NY-ESO-1 and XAGE1, respectively. After washing, an anti-NY-ESO-1 mouse monoclonal Ab (clone E978, 1:100, Invitrogen, Carlsbad, California) and USO 9-13 monoclonal Ab (2 mg/mL) were added and incubated at 4 C and room temperature overnight for NY-ESO-1 and XAGE1, respectively. After washing, sample slides were stained by the streptavidin-biotin complex (SimpleStain MAX-PO kit; Nichirei, Tokyo, Japan), followed by a reaction with 3, 3 0 -diaminobenzidine in H 2 O 2 and counterstained with hematoxylin solution.