Immunohistochemical Analysis of SOX2 Expression

The IHC assays were conducted to detect the expression profile of SOX2 in resected tumors from the sh-NC group and sh-TUG1 group, following the protocols from a previous report (23 (link)). In brief, resected tumors from the xenograft models were fixed in 3% formaldehyde overnight at 4˚C and embedded in paraffin and then cut into 5-µm sections. The slices were then incubated with 0.3% hydrogen peroxide (H₂O₂) solution in methanol for 20 min at room temperature to block the activity of endogenous peroxidases. Sections were blocked in 10% horse serum at room temperature for 1 h and then incubated with goat anti-SOX2 antibodies (1:100; cat. no. GT15098; Neuromics) at 4˚C overnight, followed by incubation with peroxidase-conjugated horse anti-goat IgG (cat. no. PI-9500-1; 1:2,000; Vector Laboratories, Inc.) at room temperature for 30 min. Immunoreactivity was measured using NovaRed peroxidase substrate (Vector Laboratories, Inc.) under an Eclipse TS100 fluorescence microscope (Nikon Corporation) at x20 magnification.