Immunofluorescence Staining of HSCs on Hydrogels

HSCs cultured on polyacrylamide hydrogels with different stiffness (0.4 kPa or 25.6 kPa) or human liver cryosections were prepared for IF staining, as previously described5 (link), 13 (link). Briefly, HSCs or human liver cryosections were fixed with 4% PFA (paraformaldehyde) for 15 minutes followed by permeabilization by 0.3% Triton X-100 for 5 minutes. After washing the cryosections with 1 x PBS three times, 5% BSA was administrated to block non-specific antibody binding sites. The cryosections were then placed in primary antibody diluted in blocking buffer and incubated overnight at 4°C. After rinsing with 1 x PBS three times, the samples were then incubated with fluorescent secondary antibody, and DAPI respectively. The samples on the slides were visualized with a Zeiss Instruments confocal microscope (Cal Zeiss AG, Jena, Germany). ImageJ was utilized to quantify the immunofluorescence density of each image.