Sox10 antibody

Total cell extracts were prepared and Western blots were performed as described (28 (link)). Antiserum to BRG1 was previously described (38 (link)). The BRM and MITF antibodies were from Abcam (Cambridge, MA, USA), the SOX10 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA), the FLAG antibody was from Sigma (St. Louis, MO, USA), BAF60A and BAF60B antibodies were from Bethyl Laboratories (Montgomery, TX, USA) and the Tubulin antibody was from Cell Signaling Technology (Boston, MA, USA).

Detailed steps for IHC were following the manufacturer’s protocol. Briefly, antigen retrieval was performed in citrate buffer (pH 6.0) for 20 minutes using a microwave oven. After blocking in 10% normal blocking serum at room temperature for 10 minutes, slides were incubated with Sox10 antibody (1:200; Santa Cruz Biotechnology Inc., Dallas, TX, USA) at 4°C overnight. Negative (omission of the primary antibody) and positive controls were included according to manufacturer’s datasheet of each antibody.
Semiquantitative assessment of the staining, including analysis of both the intensity and percentage of the stained cells, was used. The staining intensity was scored as 0, 1+, 2+, or 3+ corresponding to colorless, buff, brownish yellow, and dark brown, respectively. The percentage of positive stained cells at each intensity was estimated. Final scores ranging from 0 to 300 was calculated by multiplying the intensity and the percentage scores. The cutoff value for Sox10 was chosen based on a measure of heterogeneity using the log-rank test statistical analysis with respect to overall survival. An optimal cutoff value was identified: a staining index of ≥14 was used to define tumors of high expression and ≤13, tumors of low expression.