Alexa fluor 594 conjugated secondary antibody

The CRC DLD1 cells were evenly seeded into the confocal dishes. When the cells in the dishes grew to an appropriate density followed by washing with PBS. Then, the cells were fixed using 4% paraformaldehyde at 4°C for the overnight storage, and after permeabilization with 0.1% TritonX−100/PBS, 5% BSA/PBS was used to block for 1 h. Then, the dishes were incubated with mouse anti-α-tubulin antibody (1:100, Proteintech Group) overnight at 4°C. Samples were stored at room temperature for 0.5 h, next washed three times in PBS and incubated Alexa Fluor 594 conjugated secondary antibody (1:100, ZF−0513, ZSGB-BIO, Beijing, China), and then kept in dark place at room temperature for 1 h. Nuclear stained with DAPI for 5 min (C0065, Solarbio, Shenzhen, China). Finally, the samples were imaged by Olympus confocal microscope. In addition, permeabilization and blocking were omitted when staining with Phalloidin (1:100, Cytoskeleton, Shanghai, China). It can be directly dyed in dark at room temperature for 1 h, and then re-stained with DAPI to take photos.

Cells were plated and grown on coverslips overnight. After fixed in 4% paraformaldehyde, cells were treated with 0.5% triton X-100 for cell permeabilization. The coverslips were then immersed in blocking solution (Beyotime) for 1 h, followed by incubation with anti-p-GSK-3β (1:300, CST) and anti-p-Akt (1:200, CST) overnight. The Alexa Fluor 594-conjugated secondary antibody (1:200, Zsbio) was used for fluorescence labeling. Coverslips were incubated with DAPI for nuclei staining. Cells were observed and pictures were acquired by fluorescence microscope.

IPEC-J2 cells were cultured in a 24-well plate. Cells were treated as described for western blot analysis. The methods used for immunofluorescence staining of cells were described previously [31 (link)] and were followed with slight modifications. In brief, after 30 min of fixation with 4% paraformaldehyde solution, cells were permeabilized with 0.2% Triton X-100 for 10 min and blocked with 5% BSA for 1 h. Cells were incubated with a rabbit anti-Nrf2 antibody overnight at 4 °C and were then incubated with an Alexa Fluor 594-conjugated secondary antibody (ZF-0513, ZSGB-BIO) for 1 h. Subsequently, cell nuclei were stained with DAPI (Alexa Fluor® 555; ab150078; Abcam) for 10 min and imaged immediately using an Olympus fluorescence microscope (Tokyo, Japan). Each treatment group was analyzed in triplicate.