Mouse anti rat h3k27me3 monoclonal antibody

Cells at the exponential phase were cultured in a 24-well plate with a polylysine-coated coverslip for 24 h. Cells were incubated with mouse anti-rat H3K27me3 monoclonal antibody (1:200 dilution, Abcam) overnight at 4 °C, rinsed and incubated with goat anti-mouse fluorescent FITC-labeled secondary antibody (1:3000 dilution, Abcam) at 37 °C for 40 min. The coverslip was subjected to DAPI nuclear counterstain, treated with an anti-quenching agent, and mounted in neutral gum. The coverslip was then observed under a confocal laser fluorescence microscope. The mean number of cells with green fluorescence (target protein) in 5 randomly selected visual fields was recorded. The proportion of DAPI-positive cells indicated the relative expression of H3K27me3.

Total protein of NSCs was extracted using a protein extraction kit and quantified using a BCA kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacture’s instruction. Equal amounts of total protein (20 μg) were separated by SDS-PAGE electrophoresis and transferred to polyvinylidene difluoride membranes. The membrane was blocked in TBS buffer with 5 % skim milk and 0.1 % Tween20 for 1 h, and incubated respectively with mouse anti-rat H3K27me3 monoclonal antibody (1:200 dilution, Abcam), mouse anti-rat GAPDH monoclonal antibody (1:1000 dilution, Abcam), mouse anti-rat ACAT2 monoclonal antibody (1:1000, Abcam), and mouse anti-rat β-tubulin monoclonal antibody (1:10000, Abcam) overnight at 4 °C. The membrane was then incubated with goat anti-mouse HRP-labeled secondary antibodies (1:2000 dilution, Abcam) at 37 °C for 2 h, and subjected to ECL detection for 5 min. The intensity of bands was detected by a Molecular Imager® ChemiDocTM XRS System (Bio-Rad Laboratories). The gray value of bands was analyzed by Image Lab 2.0 software (Bio-Rad Laboratories).