Rabbit anti aldh2 antibody

CFs in each group were collected and homogenized in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) (add PMSF 0.1 mM) for 1 hour on ice. The lysates were centrifuged at 10,000 rpm and 4°C for 15 min, and the supernatants were used for Western blot after protein quantification. Proteins were separated by SDS-PAGE and blotted onto polyvinylidene fluoride (PVDF) membranes [16 (link)]. After blocking by nonfat milk for 2.5 h, immunoblotting was performed with the following antibodies: anti-GAPDH antibody (36 kDa, 1 : 1000, Boster Biological Technology, Wuhan, China) as a control for loading, rabbit anti-ALDH2 antibody (56 kDa, 1 : 3000, Abcam Co., Cambridge, UK), and 4-HNE (70 kDa, 1 : 2000, Abcam Co., Cambridge, UK). Detection and quantification were performed by ECL with horseradish peroxidase- (HRP-) linked anti-rabbit IgG (1 : 10,000, Boster Biological Technology, Wuhan, China). Densitometric quantification of antibody-specific dots was performed with ChemiDoc™ XRS+ System and analyzed with Tanon software (version 4.2.1).

ALDH2 expression of CFs was detected by double-label immunofluorescence staining technique. Anti-vimentin antibody (1 : 200, Boster Biological Technology, Wuhan, China) and rabbit anti-ALDH2 antibody (1 : 100, Abcam Co., Cambridge, UK) were used in the experiment operation. Cell slides were incubated with mixed primary antibody overnight at 4°C after blocking with 5% bovine serum albumin (BSA) for 30 min at 37°C. The mixed secondary antibodies were added and incubated in 37°C for the optimized time and dilution. The nuclei of CFs were stained with DAPI for 10 min in 37°C. Fluorescent images were obtained with fluorescence microscope camera (OLYMPUS U-HGLGPS, Japan).