Mouse anti acetylated tubulin monoclonal antibody

For protein extraction, RIPA buffer was used to lyse cells and tissue (10 mm of the spinal cord containing the injury epicenter). Protein concentrations were detected by using a Bicinchoninic acid (BCA) assay kit. The denatured proteins were electrophoretically transferred to a 10% SDS-PAGE gel and subsequently transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated at 4°C overnight with a mouse anti-acetylated tubulin monoclonal antibody (1:1,000; Proteintech, USA), rabbit anti-H3K27me3 antibody (1:1,000; CST, USA), rabbit anti-NeuroD1 antibody (1:1,000; Abcam, USA), rabbit anti-UTX antibody (1:1,000; Abcam, USA), actin (1:5,000; Abcam, USA), rabbit anti-β-tubulin antibody (1:1,000; CST, USA), and anti-GAPDH antibody (1:5,000; Abcam, USA). After the membranes were washed with 1% Tris-HCl+Tween (TBST) 3 times (15 min each), they were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000; Proteintech, USA) for 90 min. Immunoreactive bands were visualized using enhanced chemiluminescence.

After being washed with PBS three times (5 min each), cells were fixed with 4% paraformaldehyde for 15 min. After fixation, the cells were permeabilized with 0.5% PBST (Triton X-100 dissolved in PBS) at room temperature for 20 min and blocked with 4% BSA (dissolved in pure water) for 45 min. The cells were incubated with the following primary antibodies: a mouse anti-acetylated tubulin monoclonal antibody (1:200; Proteintech, USA), rabbit anti-tyrosinated tubulin polyclonal antibody (1:400; Millipore, USA), and rabbit anti-beta-III tubulin polyclonal antibody (1:400; Abcam, USA). The following day, secondary antibodies (anti-mouse immunoglobulin [IgG] antibodies or anti-rabbit IgG antibodies) were added for 1 h at 37°C.