Alexa fluor conjugated secondary antibody

Cells were immobilized in 4% paraformaldehyde for 15 min at room temperature and sealed with 5% BSA and 0.03% Triton X-100 for 50 min. After washing with PBS, the cells were incubated with primary antibody against COL1A2 (1:100 dilution) and MMP1 (1:200 dilution) at 4 °C overnight. Alexa Fluor conjugated secondary antibody (Beyotime, Nanjing, China) was incubated at 37 °C for 1.5 h, and the nucleus was stained with DAPI (Beyotime, Nanjing, China). Observations were performed using a laser confocal microscope (Zeiss LSM 800 with Airyscan).

After alcohol-soaked glass coverslips (1 cm²) were placed flat in 6-well plates, COS-1 cells (2.5 × 10⁵) were allowed to be grown on the glass-stored plates for 24 h, and then subjected to transfection with each of the indicated plasmids expressing Keap1-V5, Keap1α-V5, and Keap1β-V5, respectively. After transfection for 8 h and then recovery for 24 h, the cells were fixed with a paraformaldehyde fixative (Servicebio, Wuhan, China) for 30 min at room temperature, and then permeabilised with 0.2% Triton X-100 for 20 min. The fixed cells were incubated with 1% BSA for 60 min, and immunoblotted with each of the primary antibodies overnight, and the secondary Alexa Fluor-conjugated secondary antibody (anti-mouse or anti-rabbit antibody, ZSGB-BIO) for 4 h. The glass-mounted cells were then rinsed with PBS three times, before being subjected to DNA staining with DAPI (Beyotime, C1005). The immunofluorescent images were achieved by confocal microscopy with an IN Cell Analyzer Zeiss LSM900 cellular imaging system [40 (link)].