IF

Coverslips with adhered RPE1 cells were washed with PBS and fixed with methanol for 10 min at −20°C. Coverslips were washed with PBS two times for 2 min. Cells were permeabilized using permeabilizing buffer (PBS and 0.5% Triton X-100) for 3 min on ice. Coverslips were washed twice with PBS for 2 min. Blocking buffer (1% BSA and 0.05% Tween-20) was added for 1 h. Primary antibodies (LC3B rabbit polyclonal antibody; 1:200; NB600-1384; Novus Biologicals; LAMP2 mouse monoclonal antibody; 1:100; ab25631; Abcam) were diluted in blocking buffer,added on coverslips, and incubated overnight. Coverslips were washed three times with PBS for 5 min each. Secondary antibodies (Alexa Fluor 488-goat anti-rabbit IgG secondary antibody; A-11008; Invitrogen; Alexa Fluor 568-goat anti-mouse IgG secondary antibody; A-11004; Invitrogen) were diluted 1:500 in blocking buffer and incubated for 1 h at RT. Coverslips were washed three times with PBS for 5 min and stained with DAPI. Coverslips were mounted using Vectashield (H1000; Vector Laboratories) and sealed.