ATG5 Antibody (N-term)

Immunohistochemical analysis of paraffin-embedded human liver tissue was performed on the Leica BOND RXm. Tissue was fixed with formaldehyde at room temperature, antigen retrieval was by heat mediation with a EDTA buffer (pH9.0). Samples were incubated with primary antibody (1:500) for 1 hours at room temperature. A undiluted biotinylated CRF Anti-Polyvalent HRP Polymer antibody was used as the secondary antibody.

Fluorescent image of U251 cells stained with ATG5 (N-term) antibody. U251 cells were treated with Chloroquine (50 μM, 16 h), then fixed with 4% PFA (20 min), permeabilized with Triton X-100 (0.2%, 30 min). Cells were then incubated with ATG5 (N-term) primary antibody (1:200, 2 h at room temperature). For secondary antibody, Alexa Fluor 488 conjugated donkey anti-rabbit antibody (green) was used (1:1000, 1 h). Nuclei were counterstained with Hoechst 33342 (blue) (10 μg/ml, 5 min). ATG5 immunoreactivity is localized to autophagic vacuoles in the cytoplasm of U251 cells.

The anti-APG5L Pab is used in Western blot to detect APG5L in mouse brain tissue lysate.

All lanes: Anti-hAPG5L-D3 at 1:1000 dilution. Lane 1: SH-SY5Y whole cell lysate. Lane 2: Hela whole cell lysate. Lysates/proteins at 20 µg per lane. Secondary Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/10000 dilution. Predicted band size: 32 kDa. Blocking/Dilution buffer: 5% NFDM/TBST.