Rabbit anti human lc3 antibody

Cells were fixed in paraformaldehyde (4%) at 4°C for 10 min and permeabilized with 0.01% Triton X-100. After blocking with 10% BSA for 1 h, cells were stained with rabbit anti-human LC3 antibody (Cell Signaling Technology, BA) overnight at 4°C. After washing, cells were incubated with secondary anti-rabbit IgG Alexa 488 for 1 h in the dark. Unbound antibody was removed with PBS, cells were analyzed using a confocal microscopy. To study the co-localization of LC3 and p62, rabbit anti-LC3 and rabbit anti-p62 (Santa Cruz, 1:100) antibodies were used. First, cells were stained with rabbit anti-LC3 antibody and secondary anti-rabbit IgG Alexa-488. Cells were then stained with rabbit anti-p62 antibody following treatment with 10% BSA for 1 h. After washing with PBS, anti-rabbit IgG Alexa-568 secondary antibody was added for 1 h and analyzed using a confocal microscopy.

For immunohistochemistry, the slides were incubated with antibody against LC3 (1:100, Cell Signaling Technology) at 4°C overnight and stained with diaminobenzidine (DAB) and counterstained with hematoxylin. The slides were then subjected to gradient ethanol dehydration and dimethyl benzene transparent and mounted with neutral resin cover slides. Images were captured using a Nikon (Tokyo, Japan) ECLIPSE 80i.
For immunofluorescence staining, cells cultured on poly-l-lysine-coated cover glasses were washed by PBS once and then fixed with 4% paraformaldehyde for 15 min at room temperature. After washing, the cells were permeabilized by 0.1% Triton X-100 for 15 min at room temperature. Next, the cells were blocked with 2% BSA for 30 min and then stained with rabbit anti-human LC3 antibody (1:200, Cell Signaling Technology) at 4°C for 6 h. After washing by PBS, the cells were probed with Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:200, Thermo Fisher Scientific) at 4°C for 1 h. To display the cell nuclei, 100 μg/mL DAPI was applied to incubate with cells for 15 min at room temperature. After washing, the cells were subjected to an inverted fluorescence microscope (Olympus IX73, Olympus, Tokyo, Japan) for imaging. The LC3 puncta were measured by ImageJ software (NIH). Five individual pictures were analyzed for each kind of cell.