Anti rabbit igg tritc

IF staining was performed according to the standard protocol (Santa Cruz Biotechnology). Briefly, a 5-μm frozen section from each sample was fixed in acetone at 4 °C overnight. After washing with PBS three times, sections were blocked with 1% bovine serum albumin for 30 min and incubated overnight at 4 °C with rabbit anti-human LC3, SQSTM1, ATG7 or LAMP1 primary antibody (1 : 50). Following PBS washes, the sections were incubated with anti-rabbit IgG–TRITC (1 : 100, Santa Cruz Biotechnology) for 2 h at room temperature in the dark, and then washed again with PBS. Nuclei were stained with diamino-phenyl-indole (DAPI, 1 μg/ml; Sigma-Aldrich, St Louis, MO, USA) for 5 min at room temperature. Coverslips were mounted with SlowFade Gold Antifade Reagent (Invitrogen, Carlsbad, CA, USA), and all sections were imaged using a laser confocal microscope (Olympus, Tokyo, Japan).

Transfected or control OVCAR3 cell slides were fixed with 4% formaldehyde, blocked using 5% skimmed milk in 0.01 M PBS, pH 7.2 (PBS-M). Primary antibodies targeting LC3, SQSTM1 (P62) or ATG3 (Sigma-Aldrich, St Louis, MO, USA), were diluted 1:500 in PBS and added to the slides. After incubation overnight at 4°C, slides were washed once with 0.2% Tween 20 in PBS and twice with PBS, and then incubated for 30 min at room temperature with anti-rabbit IgG–TRITC (1:100, Santa Cruz Biotechnology). The slides were washed three times with PBS, then fixed with glycerol/PBS (2:1, pH 9.0), and observed and photographed using a laser confocal microscope (Olympus, Tokyo, Japan).