Anti sox2 antibody

Chromatin immunoprecipitation (ChIP-PCR) was carried out using Simple ChIP plus Enzymatic Chromatin IP Kit (Cell Signaling: #9005) following the manufacturer’s instructions. In summary, 5 × 10⁶ cells were plated in a 100 mm culture dish with 7 mL of complete FBM and cultured for 16 h. Following whole-cell cross-linking with 1% formaldehyde and lysis, the nuclear pellet was digested by Micrococcal nuclease and sonicated to achieve desired chromatin fragmentation. The resulting chromatin−protein complexes were incubated with anti-SOX2 antibody (Cell Signaling Technology) or negative control IgG (Vector Laboratories) overnight. After immunoprecipitation, protein G magnetic beads (50 μL) were added and incubated and the antibody complexes were incubated at 65 °C overnight for reverse cross-linking. The purified DNA was then collected, and the relative enrichment of specific regions in ChIP-DNA was determined using SYBR Green Master Mix (Applied Biosystems) in an ABI 7500 Real-Time PCR (Applied Biosystems). Primer oligonucleotides used in the ChIP study are shown in the Supporting Information, Table S3.

Five micron sections were stained with anti-SOX2 antibody (Cell Signaling Technologies #23064) at a dilution of 1:300. Biotinylated goat anti-rabbit antibody (diluted 1:100), was applied and incubated for 30 minutes. Slides were incubated with Avidin-Biotinylated HRP Complex (ABC reagent) for 30 minutes, followed by a 5-minute incubation with the HRP substrate, DAB (3,3’-diamnobenzidine) from the Vectastain ABC HPR kit (Cat. PK-4001, Vector Laboratories, Burlingame, CA). SOX2 staining densities within cancer cores were quantified using Aperio ImageScope by an experimenter naïve to clinical information. The immunohistochemical score of a given specimen was averaged from all optimally stained cores. Cores with inadequate representation of tumor tissue were omitted from further analysis. H-Scores were determined using the nuclear quantification algorithm according to the following formula: H score = [(3×strong%)+(2×moderate%)+(weak%)]. Continuous variable scoring was used to evaluate correlations with other markers but we also looked at the dichotomous versions for SOX2, but not e-cadherin or vimentin. Low expression of SOX2 was defined as scores of 0 or 1. SOX2 expression scores were available on 195 patients.