Goat anti mouse igg h l alexa fluor plus 555

Forty-eight hours after transfection, cells were seeded on glass coverslips and then fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and blocked for 1 h in 2% BSA. Immunostaining was conducted with mouse anti-actin antibody (1 : 1000, ProteinTech) and rabbit anti-Flag antibody (1 : 200, ProteinTech) overnight at 4°C. Goat anti-mouse IgG (H+L) Alexa Fluor Plus 555 and goat anti-rabbit IgG (H+L) Alexa Fluor Plus 488 secondary antibodies (Invitrogen, USA) were used at 1 : 1000 at room temperature for 1 h. The cell nuclei were stained with DAPI (6-diamidino-2-phenylindole). Protein localization was observed by fluorescence microscopy (Carl Zeiss, Germany).

The AGS cells were fixed with 4% paraformaldehyde for 10 min and permeabilized 0.2% Triton X-100 (T8200, Solarbio) for 10 min at room temperature. Subsequently, the cells were blocked with PBS supplemented with 5% BSA (ST025, Beyotime) for 1 h. AGS cells were incubated overnight at 4 °C with rabbit anti-USP12 polyclonal antibody (PA5-139844, Invitrogen) and mouse anti-YAP antibody (SC-110199, Santa Cruz). Then the following day, cells were incubated at room temperature for 1 h using Goat anti-Mouse IgG (H + L) Alexa Fluor™ Plus 555 (Invitrogen, 32727) for mouse antibody and Goat anti-Rabbit IgG (H + L) Alexa Fluor™ Plus 488 (Invitrogen, 32731) for rabbit antibody. Three washes later, the cells were stained with DAPI (Beyotime, C1005) to visualize the cell nucleus. The images were captured with a confocal laser scanner and further processed and analyzed using ImageJ.