Fluorescent image of U251 cells stained with ATG4D antibody. U251 cells were treated with Chloroquine (50 mu M,16h), then fixed with 4% PFA (20 min), permeabilized with Triton X-100 (0.2%, 30 min). Cells were then incubated ATG4D 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, 1h). Cytoplasmic actin was counterstained with Alexa Fluor 555 (red) conjugated Phalloidin (5.25 mu M, 25 min). Nuclei were counterstained with Hoechst 33342 (blue) (10 ug/ml, 5 min). Pictures were taken on a Biorevo microscope (BZ-900, Keyence). ATG4D immunoreactivity is localized to autophagic vacuoles in the cytoplasm of U251 cells.