Alexa 488 and alexa 568 fluorescent secondary antibodies

Cells (5 × 10⁴ cells/35-mm dish) were exposed to 50 ng/mL of NGF for 72 h, rinsed with 1X PBS 3 times for 10 min each and then fixed with 4% paraformaldehyde (PFA) for 10 min. The fixed cells were rinsed with 1× PBS 3 times for 10 min each and then permeabilized with 1× PBS containing 0.1% Triton-X100 for 10 min at RT. The cells were then blocked with 1× PBS containing 1% BSA and 5% goat serum for 1 h at RT after being rinsed with 1× PBS 3 times for 10 min each. After blocking, the cells were incubated with the following primary antibodies in 1% BSA and 5% goat serum in 1× PBS overnight at 4 °C: anti-calsenilin (anti-1F11 or anti-4E4) [21 (link)] and anti-RhoA (SantaCruz). The cells were subsequently incubated with either Alexa Fluor 568 goat anti-rabbit IgG or Alexa Fluor 488 goat anti-mouse IgG antibodies (Invitrogen) for 1 h, and then they were incubated with Alexa 488 and Alexa 568 fluorescent secondary antibodies (Thermo Fisher Scientific) for 1 h. After being rinsed with PBS, the cells were mounted in 4′,6-diamidino-2-phenylindole (DAPI)-containing Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA, USA) to label the nuclei and visualized using a confocal laser scanning microscope (LSM 700; Carl Zeiss, Oberkochen, Germany).

ZW13-2 cells (5 × 10⁴ cells/35-mm dish) were rinsed with 1× PBS 3 times for 10 min each and then fixed with 4% paraformaldehyde (PFA) for 10 min. The fixed cells were rinsed with 1× PBS 3 times for 10 min each and then permeabilized with 1× PBS containing 0.1% Triton-X 100 for 10 min at RT, and then cells were blocked with 1× PBS containing 1% BSA and 5% goat serum for 1 h at RT after being rinsed with 1× PBS 3 times for 10 min each. After the cells were blocked, they were incubated with the following primary antibodies in 1% BSA and 5% goat serum in 1× PBS overnight at 4 °C: anti-Cx43 (Abcam) and anti-RhoA (Santa Cruz). Cells were subsequently incubated with either Alexa Fluor 488 goat anti-mouse IgG antibodies (Invitrogen) or Alexa Fluor 568 goat anti-rabbit IgG for 1 h and then incubated with Alexa 488 and Alexa 568 fluorescent secondary antibodies (Thermo Fisher Scientific) for 1 h. Control reactions omitting the primary antibodies resulted in no labeling with the secondary antibodies (data not shown). After being rinsed with PBS, cells were mounted in 4′,6-diamidino-2-phenylindole (DAPI)-containing Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) to label the nuclei and visualized using an LSM700 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).