SY5Y cells were pretreated with 5nM bafilomycin for 24hr and fixed in methanol (left panel) or 4% of paraformaldehyde (right panel). Treatment with antibody at dilution 1:100. Data courtesy of Jianhui Zhu, MD, PhD & Charleen T. Chu, MD, PhD, University of Pittsburgh School of Medicine.
Time course study of mouse leukemic monocyte macrophage cells treated with U18666A, a drug that causes cholesterol and lipid storage in cells, thereby blocking fusion between late endosomes and lysosomes. Cleaved-LC3 (APG8b) antibody detected punctuate staining indicative of autophagic vacuole or phagosome structures. Data courtesy of Dr. Barry Boland, Department of Pharmacology, Oxford University.
Fluorescent image of U251 cells stained with cleaved LC3B 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 cleaved LC3B primary antibody (1:100, 2 h at room temperature). For secondary antibody, Alexa Fluor 488 conjugated donkey anti-rabbit antibody (green) was used (1:1000, 1h). Nuclei were counterstained with Hoechst 33342 (blue) (10 ug/ml, 5 min). LC3 immunoreactivity is localized to autophagic vacuoles in the cytoplasm of U251 cells.