Gt100540

The PD-L1 expression in each patient was determined by immunohistochemistry. The tumor tissue slides were baked at 63°C for 60 min and then boiled in 10 mM sodium citrate (pH 6.0) for 30 min to retrieve the antigen. Anti-PD-L1 polyclonal antibody was used as the primary antibody (Abcam, Cambridge, United Kingdom; rabbit SP142, 1:300; Dako IHC 22C3, 1:300) and goat anti-rabbit biotinylated IgG as the secondary antibody (Maxim, UltraSensitive^TM SP IHC Kit, KIT-9707). The slides were then counterstained with hematoxylin (Gene, GT100540), dehydrated, and mounted. Images were digitally scanned at ×20 magnification.

Immunohistochemistry studies of IER5 were performed on glioma samples in a TMA. The human glioma TMA (Product number: HBraG180Su01) was purchased from Shanghai Outdo Biotech Co., Ltd. (Shanghai, China). Ethical approval was granted by ethics committee of Shanghai Outdo Biotech Company. The 180 cases of glioma in this microarray were from Chinese National Human Genetic Resources Sharing Service Platform⁴. And the platform number is 2005DKA21300. In brief, all the samples were incubated with an anti-IER5 antibody (Invitrogen, PA5-56287, United States; 1:300 dilution) overnight at 4°C. Afterward, the samples were incubated with HRP-labeled secondary antibodies for 1 h at 37°C. Finally, the samples were stained and imaged. EnVision Plus System-HRP (K8002, DAKO, Denmark) was employed, and IER5 was visualized with diaminobenzidine (DAB) as the substrate. The nucleus was stained with Mayer’s hematoxylin counterstain (GT100540, Gene). The assessment of IHC data was carried out by two readers and verified by two independent pathologists blinded to the clinicopathologic information. The staining intensity was defined as: 3 (strong), 2 (moderate), 1 (weak), and 0 (negative). Negative and weak staining intensity was classified as low IER5 expression, whereas strong and moderate staining intensity was classified as high IER5 expression.