Paraformaldehyde phosphate buffer

Transfected COS-7 cells were fixed with a 4% paraformaldehyde phosphate buffer (Nacalai Tesque) for 15 min at room temperature and permeabilized by 0.1% Triton X-100 in PBS for 10 min at room temperature. The fixed cells were stained by a Phalloidin-iFluor 633 working solution (Abcam, ab176758) for 1 h. After three washes in PBS, mounting media ProLong (Thermo Fisher) was added.

For immunofluorescence analysis, untransfected MIN6 cells or cells transfected using Polyethyleneimine max were washed with PBS and fixed with 4% paraformaldehyde phosphate buffer (Nacalai Tesque) on ice for 4.5 min. Fixed cells were washed with PBS and permeabilized by incubation on ice for 4.5 min in PBS containing 0.2% Triton X-100. After incubation in PBS containing 3% fetal bovine serum and the primary antibody for 2 hr at room temperature, cells were incubated with secondary antibodies labeled with Alexa Fluors 488, 568, or 633 (Thermo Fisher Scientific) for 1 hr at room temperature. Images were acquired at room temperature at 100 × magnification using a DM IRE2 and confocal software (both from Leica).