Alexa fluor 488 546 conjugated secondary antibody

The colocalization of IP3R and VDAC1 and the expression of calpain-1 were examined by the indirect immunofluorescence method. The cells were cultured on coverslips overnight, then treated with the indicated drugs for 24 h and rinsed with 0.1 M PBS three times. After incubation, the cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 5 min, washed three times with 0.01 M phosphate-buffered saline (PBS) and then blocked for 30 min in 5% (w/v) non-immune animal serum (goat; Beyotime Institute of Biotechnology, Shanghai, China) PBS and incubated with primary antibodies (IP3R, VDAC1 and PDI) overnight at 4°C. The following day, the slides were incubated with the Alexa Fluor-488/546-conjugated secondary antibody (1:400 dilution; Invitrogen Life Technologies) for 1 h, and then stained with Hoechst 33342 (2 µg/ml) for 2 min and washed three times with PBS. After mounting, the cells were examined by Olympus FV1000 (Olympus, Tokyo, Japan) confocal laser microscopy.