Ab243041

Immunohistochemistry (IHC) was performed on 4-μm-thick paraffin sections of human tissues using standard protocols with optimized conditions. Tissue samples for IHC assays were obtained from a cohort of 47 RB patients who had undergone treatment at the Zhongshan Ophthalmic Center. All RB samples were reviewed by specialized pathologists. The expression of SOX4 (Abcam, ab243041, 1:1000) was evaluated with the HRP-conjugated Goat anti-mouse IgG (H + L) (Servicebio, GB23301, 1:200) using semi-quantitative methods that considered both the proportion and intensity of stained tumor cells. The percentage of positively stained cells was counted in five randomly selected fields under a light microscope (×400), and staining intensity was graded as follows: negative (0), weak (1+), moderate (2+), or intense (3+). The percentage of stained cells was categorized as follows: ≤10% (grade 1), 11–50% (grade 2), or >50% (grade 3). The final IHC scores were calculated by multiplying the scores for the percentage of stained cells and staining intensity. In this study, the highest score obtained was 9, while the lowest was 0. SOX4 expression was deemed high when the IHC score was ≥ 4 and low when the IHC score was < 4. The significance of the difference was evaluated using Fisher’s exact test.

The RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) was supplemented to the cells and the total protein was extracted on the ice. After the samples were centrifuged, the supernatant was collected as the protein samples. BCA method was applied for protein quantification. Then, 5× loading buffer was mixed with the samples, and next the protein was denatured in boiling water. After SDS-PAGE, the protein was transferred on the PVDF membrane, and then the membrane was blocked in 3% bovine bull serum albumin (BSA) for 1 h. Then, the membranes were incubated with the primary antibodies including anti-SOX4 (ab243041, 1:1000, Abcam, Shanghai, China) and anti-GAPDH (ab8245, 1:3000, Abcam, Shanghai, China), respectively, overnight at 4°C. After the residual primary antibody on the membrane was rinsed off by TBST on a shaker, the membrane was incubated with HRP-labeled secondary antibody (goat anti-mouse IgG H&L, ab205719, 1:2500, Abcam, Shanghai, China) for 1 h at room temperature. After the residual secondary antibody on the membrane was washed off by TBST on the shaker, the Enhanced Chemiluminescence Western blotting Substrate (Dongguan Biotech, Shandong, China) was added on to the membrane to react with the protein-antibody complex, and the protein bands were developed. GAPDH was the internal reference.