Fluorescein isothiocyanate labeled goat anti mouse secondary antibody

GBC-SD cells at the logarithmic growth phase were seeded in 24-well plates at a density of 1×10⁷ cells/well, fixed with 4% paraformaldehyde for 20 min at room temperature, blocked with 200 µl 1% bovine serum albumin (Beyotime Institute of Biotechnology, Haimen, China) for 30 min at room temperature, and incubated with mouse anti-human CD133 monoclonal antibody (dilution, 1:11; cat no. 130-050-801; Miltenyi Biotec GmbH) at 4°C overnight. PBS was used as a negative control. Subsequently, fluorescein isothiocyanate-labeled goat anti-mouse secondary antibody (dilution, 1:200; cat no. 115-095-003; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) was added at 4°C for 1 h. Cell nuclei were stained with DAPI at 4°C for 1 h. Cells were subsequently observed (9 non-overlapping fields of view) using a Nikon ECLIPSE Ti-S fluorescence microscope (Nikon Corporation, Tokyo, Japan).

Immunofluorescence (IF) staining was performed to visualize ER state. Briefly, HK‐2 cells were washed by PBS, fixed by 4% formaldehyde for 15 minutes and permeabilized for 10 minutes with 0.2% Triton X‐100. After blocking nonspecific binding with 5% BSA for 30 minutes, staining with ER stress marker protein disulfide‐isomerase (PDI) was performed by incubating with a mouse monoclonal antibody against PDI (ThermoFisher, CA) for 1.5 hour at RT, thereafter incubated with the fluorescein isothiocyanate‐labeled goat anti‐mouse secondary antibody (Jackson, West Grove, PA) for 2 hours at RT. Fluorescence‐labeled cells were mounted with Vectashield Mounting Medium plus 4′,6‐diamidino‐2‐phenylindole (Vector Laboratories, Burlingame, CA), followed by visualizing under a fluorescence microscope (Olympus, Tokyo, Japan) and analysis by ImageJ software (http://rsb.info.nih.gov/ij) in 20 randomly selected fields for each coverslip at ×400 magnification.