Sox9 antibody

A ChIP assay was performed according to the manufacturer’s instructions (SimpleChIP Plus Sonication Chromatin IP Kit, CST).
An HCT116^CD133+CD44+ cell pellet was subjected to cross-linking, cross-linking suspension, cell and nuclear lysis, ultrasonic disruption, and chromatin dilution. The lysate was immunoprecipitated with Sox9 antibody (Abcam, USA). Then, the purified DNA was quantitatively pulled down using qPCR. The sequences of the Cldn7 primers were 5’-TGTTGGGAAGAAAGGAAGG-3’ (forward primer) and 5’-CCAGGTGAGGAGGAAGAA-3’ (reverse primer).

The paraffin-embedded tissues were cut to 3-μm sections. Dako Envision FLEX+ system (K8012; Dako, Glostrup, Denmark) was used to deparaffinized and epitopes were unmasked in PT link with low pH target retrieval solution (Dako, Denmark). Briefly, the slides were blocked for 5 minutes with peroxidase blocking solution (Dako) at room temperature (RT), and then incubated with rabbit polyclonal Ki67 antibody (cat. no. ab15580; 1:800; Abcam, USA), rabbit polyclonal p53 antibody (cat. no. ab131442; 1:500; Abcam, USA) and rabbit polyclonal sox9 antibody (cat. no. ab26414; 1:1000; Abcam, USA) at 4°C overnight before incubated with rabbit linker (Dako, Denmark) for 15 minutes, horseradish peroxidase (Dako) for 30 minutes at RT. The slides were subsequently stained with 3,3′-diaminobenzidine tetrahydrochloride for 10 minutes, counter-stained with hematoxylin, dehydrated, and mounted in Richard-Allan Scientific Cytoseal XYL (Thermo Fisher Scientific, Waltham, MA, USA) before evaluated under an optical double-headed microscope (Olympus, Tokyo, Japan).

For immunohistochemistry (IHC), 8 μm sections of formalin-fixed and paraffin-embedded brain tissues were first de-waxed and rehydrated before antigen retrieval. The TGF-β1-antibody (1:100 dilution; Proteintech, China) and Sox9-antibody (1:250 dilution; Abcam, ab76997) were used for this study. After incubation with the primary antibodies, the tissues were rinsed and incubated for 1 h with Biotin-labeled secondary antibodies at room temperature (Molecular Probes 1:800). Nuclei were stained by Hematoxylin. Stained sections were examined under a light microscope and the positive cells in five high power fields (1 × 400) were counted for statistic study. The relative expression of TGF-β1 and Sox9 was analyzed by Graphpad via Spearman rank correlation test.