IHC Quantification of OSR1 Expression

After routine deparaffinizing, rehydrating, hydrogen peroxide blocking, and retrieving tissue antigen with a microwave, the sections were incubated with rabbit polyclonal antibody to OSR1 (A18272, 1:50; Abclonal, China) at 4 °C overnight. They were stained with goat anti-rabbit IgG (H + L) HRP (31460, 1:500; Thermo Fisher Scientific, US). Diaminobenzidine solution (DA1010, Solarbio, China) was employed to counterstain, and the sections were subsequently treated with hematoxylin (H8070, Solarbio) for 1 min and dehydrated. Two pathologists, unaware of the clinical context, evaluated the immunostained tissue sections. One hundred cells were counted in each of the five view fields on each slide at 400 × magnification. The intensity of IHC staining was visualized and scored as 3 (strong stain), 2 (medium), 1 (weak), and 0 (no stain). The extent of staining ranged from 0 to 4, corresponding to the immune-reactive tumor cells percentage (76–100%, 51–75%, 26–50%, 1–25%, 0%), with 4 being the highest. Based on the staining intensity and staining scores, each sample was assigned a score between 0 and 12, categorizing it into two categories: OSR1 low expression (0–6) and OSR1 high expression (8–12) [22 (link)].