Multiplex Immunohistochemical Analysis of Tumor Microenvironment

Tissue samples were fixed for at least 48 h in 10% neutral buffered formalin, processed routinely, embedded in paraffin, sectioned 3 μm thick, mounted on glass slides, and stained with hematoxylin and eosin (HE). All tumors were examined for KDM2B expression and the presence of lymphocytes and macrophages using immunohistochemistry (IHC). IHC was performed using antibodies to Iba1 (1:2000, Fujifilm Wako, Osaka, Japan, 019-19741), CD3 (1:1000; Agilent Technologies, CA, USA, IR503), KDM2B (1:50; Santa Cruz Biotechnology, Inc.,sc-293279), CD31 (1:250; Abcam, JC/70A), PD-L1 (1:100; clone 6C11-3A11), and CD204 (1:800; Medicinal Chemistry Pharmaceutical Co., Ltd., Sapporo, Japan, KT022) as described previously^{7 (link)}. Nano Zoomer 2.0-RS (Hamamatsu Photonics, Hamamatsu, Japan) was used to scan histological slides, which were then processed in QuPath ver 0.2.1^{34 (link)}. Scanned slides were opened in QuPath as Brightfield (H-DAB), and the Estimate Stain Vectors feature was used to automatically adjust the staining colors. Normal endothelial and tumor cells were detected using the Cell Detection function. The Create Detection Classifiers function was used to label cells based on their morphologies and locations, allowing the QuPath software to correctly classify each cell type. The collected data was exported and used for further analysis.