Immunofluorescence Analysis of Protein Localization

Cells were fixed with 4% PFA (Thermo Fisher Scientific, Waltham, MA, USA) for 15 min at RT and then permeabilized with 0.1% TritonX-100 in sterile PBS for 10 min at RT, after blocking the samples with 5% BSA and 5% FBS in sterile PBS for 1 h at RT. Next, cells were incubated with the appropriate primary antibody overnight at 4 °C, and after several washing steps, secondary antibodies were used. The cell nuclei were stained with DAPI (Thermo Fisher Scientific). A Zeiss LSM-710 confocal microscopy system (Carl Zeiss Microscopy GmbH, Jena, Germany) was used to detect proteins of interest with a 63× objective. Images were analyzed with ZEN 3.2 (Carl Zeiss Microscopy GmbH, Jena, Germany) and Image J software (Version 1.53t) [38 (link)]. Primary antibodies were the same as we used for Western blot. Secondary antibodies used were Goat-Anti-Mouse antibody, Alexa Fluor 488, Thermo Fisher Scientific, Waltham, MA, USA; Cat# A-11029, Goat anti-Rabbit IgG, Alexa Fluor 488, Thermo Fisher Scientific, Waltham, MA, USA; Cat# A-11008, Goat-Anti-Rabbit antibody, Alexa Fluor 546, and Thermo Fisher Scientific, Waltham, MA, USA; Cat# A11035 for ICC analysis. Actin filaments were stained with CF^®543 Phalloidin (Biotium, Fremont, CA, USA; Cat: 00043). The colocalization coefficient was calculated with Image J-Ezcolocalization plugin according to this article [40 (link)].