Orthophotomicroscope

All GBM tissues were fixed with 4% paraformaldehyde (Cat no. 30,525–89-4, Sigma-Aldrich, USA). The tissues were then embedded in paraffin and cut into 4 μm thick sections. After heating, the tissues were deparaffinized and rehydrated using graded xylene and ethanol. After antigen retrieval using sodium citrate (Servicebio, Wuhan, China), the endogenous peroxidases in the tissue sections were blocked using hydrogen peroxide (H₂O₂). Next, 5% bovine serum albumin (Wuhan Boster Biological Technology Ltd.) was added to block nonspecific binding. The sections were then incubated with the following primary antibodies overnight at 4°C: LHX5 (1:20 dilution; cat no: ab187975; Abcam, USA) and TLX1 (1:50 dilution; cat no: 26,877-1-AP; Proteintech, Wuhan, China). Subsequently, the sections were incubated for 2 h with anti-mouse and anti-rabbit horseradish peroxidase (HRP)-conjugated goat secondary antibodies (Servicebio, Wuhan, China). The sections were then stained with 3,3ʹ-diaminobenzidine (DAB) and hematoxylin (ZSGB-BIO, Beijing, China), and images were obtained using an orthophoto microscope (Version: BX53; Olympus, Japan). Protein levels of the targets were calculated based on the product of the intensity scores (0, no staining; 1, +; 2, ++; 3, +++) and percentage of positive cells (0, 0–1%; 1, 1–33%; 2, 34–66%; 3, 67–100%).