Alexa fluor 488 tagged donkey anti mouse igg

SW480 cells were fixed on glass coverslips in 4% paraformaldehyde for 10 min at room temperature, permeabilized with 0.2% Triton X-100, and incubated with anti-ANO9 antibody (dilution 1:200, LS-C179041, LifeSpan BioSciences, Inc.) overnight at 4°C. The coverslips were washed and incubated with the Alexa Fluor 488-tagged donkey-anti-mouse IgG (1:1000, Molecular Probes, CA). For nuclear staining, SW480 cells and HEK293T cells were incubated with Hoechst 33342 (H3570, Thermofisher Scientific, 1:2,000) after ANO9 immunostaining For HA staining, HEK293T cells transfected with the HA-tagged Ano9 mutants were incubated with anti-HA antibody (1:100, Cat# H6905, Sigma-Aldrich) for 1 hr at room temperature. The cells were then washed three times and incubated with the Alexa Fluor 488 goat-anti-rabbit IgG (1:200, Life Technologies Cat# A11008) for 30 min at room temperature. Finally, the cells were washed and fixed with 4% PFA. For permeabilized staining, cells were fixed with 4% PFA and treated with 0.2% Triton 100 for 10 min. Then, the cells were incubated with the anti-HA antibody (1:100) for 1 hr, washed three times, and then incubated with the Alexa Fluor 488 goat-anti-rabbit IgG for 30 min at room temperature. After washing, the coverslips were mounted and imaged using a confocal microscopy.