Nanogold conjugated anti mouse igg antibody

HCT116 WT and TBC1D15−/− cells stably expressing YFP-LC3 and mCherry-Parkin were treated with Valinomycin for 3 hr and fixed for 30 min with 4% paraformaldehyde and 0.1% glutaraldehyde in PBS. The fixed cells were washed four times with PBS, followed by permeabilization for 40 min with 0.1% Saponin and 5% goat serum in PBS. The cells were then incubated for 1 hr with mouse anti-GFP antibody (Invitrogen clone 3E6), followed by 1 hr with nanogold-conjugated anti-mouse IgG antibody (Nanoprobes) and further processing as described (Tanner et al., 1996 (link)). Thin sections (∼80 nm) were counter stained with uranyl acetate and lead citrate. The sections were examined with a JEOL 200 CX transmission electron microscope. Images were collected with a digital CCD camera (AMT XR-100; Danvers, MA).

TBC1D15/17 DKO HCT116 cells stably expressing YFP-RAB7A and mCherry-Parkin were treated with or without valinomycin for 3 hr and then fixed for 30 min with 4% paraformaldehyde and 0.1% glutaraldehyde in PBS. The fixed cells were washed four times with PBS, followed by permeabilization for 40 min with 0.1% Saponin and 5% goat serum in PBS. The cells were incubated for 1 hr with mouse anti-GFP antibody (A-11120; Invitrogen), followed by 1 hr with nanogold-conjugated anti-mouse IgG antibody (Nanoprobes, Yaphank, NY) and further processing as described previously (Tanner et al., 1996 (link)). Thin sections (∼80 nm) were counter stained with uranyl acetate and lead citrate. The sections were examined with a JEOL 200 CX transmission electron microscope. Images were collected with a digital CCD camera (AMT XR-100; Danvers, MA).