Linc-RA1 expression detection in glioma

The expression of linc-RA1 in clinical glioma specimens was detected using ISH, performed as previously described^{49 (link)}. The sections were deparaffinized with xylene, rehydrated in serial dilutions of ethanol, and treated with 0.2 N HCL. After washing for 3 times, the sections were incubated in proteinase K (40 µg/mL, Promega) for 20 min and fixed with 4% paraformaldehyde for 10 min. The sections were reconstituted using hybridization solution and incubated at 56 °C overnight in a digoxigenin-labeled linc-RA1 probe (Exiqon, Vedbaek, Denmark). After washing, the sections were blocked with 5% normal goat serum for 1 h at room temperature followed by incubation in an anti-digoxigenin alkaline phosphatase conjugate (Roche, Stockholm, Sweden) overnight at 4 °C. Colorimetric signals were obtained by incubating the sections in 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro-blue tetrazolium chloride (NBT) buffer in the dark for 4 h at room temperature. Nuclear fast red was used as the counterstain.