Hbrag180su01

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.

U87, U251, U373, and SHG-44 cell lines were purchased from BeNa Technology (China). U87, SHG-44, and U251 were cultured in 90% DMEM-H with 10% fetal bovine serum (FBS) and U373 was cultured in 90% RPMI-1640 with 10% FBS.
Formalin-fixed glioma tissues and normal tissue chip were obtained from Shanghai Outdo Biotech Company (# HBraG180Su01). Tissues were collected from 2009.2 to 2017.7. All patients were completely informed before the collection of the tissue samples and written informed consent was provided as well.