Alexa fluor 647 anti mouse antibodies

SW480 cells were seeded on cell slides in a 24-well plate and cultured for 24 h. After the medium was decanted, the cells were washed three times with cold PBS. Then, the cells were fixed in 4% paraformaldehyde for 15 min and permeabilized in 0.5% Triton X-100 for 5 min. After the cells were washed three times with cold PBS, PBS containing 5% bovine serum albumin was used to block the samples for 1 h at room temperature. Then, 1:100 diluted indicated antibody in PBS was added to cover the cell slides and incubated overnight at 4ºC. After three washes with PBS, a 1:500 dilution of Alexa Fluor 488 anti-rabbit and a 1:1000 dilution of Alexa Fluor 647 anti-mouse antibodies (Cell Signal Technology) were added to cover the cell slides. Then, the cell nuclei were stained with DAPI for 1 h at room temperature. After five times wash with PBS, the cells on the slides were mounted by using ProLong Gold antifade reagent (Invitrogen), and images were acquired using a fluorescence microscope (Zeiss, Oberkochen, Germany).

We grew HepG2 cells on cell slides inside a 24-well plate for 24 h. The medium was then decanted and the wells were washed three times with cold PBS. The cells were then fixed in 4% paraformaldehyde for 15 min and permeabilized in 0.5% Triton X-100 for 5 min. After washing three times with PBS, the cells were blocked for 1 h at 25 °C in PBS with 5% bovine serum albumin. The primary antibodies were diluted by 1:100 in PBS with 1% bovine serum albumin (antibody dilution buffer) and incubated overnight at 4 °C. After washing three times with PBS, Alexa Fluor 488 anti-rabbit and Alexa Fluor 647 anti-mouse antibodies (Cell Signaling, USA) were added to the antibody dilution buffer at 1:500 and 1:1,000 dilutions, respectively. We then added DAPI to the slides, and incubated them for 1 h at room temperature. After washing the slides five times with PBS, we mounted them using ProLong Gold antifade reagent (Invitrogen, USA). We acquired images using a Two-photon super-resolution point scanning confocal microscope (Nikon, Japan) and selected representative images for each sample.