Fig. 2. Immunofluorescence microscopy of ApoB. (A) Labeling of ApoB with four monoclonal antibodies and one polyclonal antibody. Huh7 cells treated with 10 µM ALLN for 12 hours were fixed and permeabilized with 0.01% digitonin for 30 minutes. ApoB (red) and LD (green) are doubly labeled. One monoclonal antibody (6H12) labeled only the ApoB-crescent, whereas the other four antibodies labeled both ApoB-crescents and lysosomes (Ohsaki et al., 2006a) but did not label the ER. Bars, 10 µm. (B) Labeling of ApoB in ALLN-treated Huh7 cells that were fixed and permeabilized with 0.1% Triton X-100 for 5 minutes before labeling. One monoclonal antibody (6H12) exclusively labeled the ApoB-crescent, whereas the other four antibodies also labeled the ER network in addition to the ApoB-crescent. Bars, 10 µm. (C) Huh7 cells treated with 10 µM ALLN for 12 hours were fixed, permeabilized with 0.01% digitonin for 30 minutes, and subjected to triple labeling for ER proteins (red), ApoB (green), and LD (blue). PDI, calnexin, and transferrin showed a marked colocalization with ApoB around LDs (arrowheads). ApoB was labeled by either a goat polyclonal or mouse monoclonal (6H12) antibody. The nuclear membrane was also labeled for the ER proteins. Bars, 5 µm. (D) Huh7 cells incubated with 10 µM ALLN for 12 hours were fixed and permeabilized with 0.1% Triton X-100 for 5 minutes and labeled by PDI (red) and ApoB (green; goat polyclonal), and LD (blue). PDI was found in a network pattern throughout the cytoplasm, and labeling around LDs was not conspicuous. Bars, 5 µm.