Anti sm α actin antibody conjugated with cy3

Immunofluorescence staining was performed as described^{15 (link)}. Briefly, HSCs were washed with PBS twice and fixed in 4% buffered formaldehyde solution for 15 minutes. Following exposure to Triton X100 (0.5% in PBS) for 5 minutes and then with 3% BSA in PBS for 30 minutes, cells were incubated with primary antibodies at room temperature for 1 hour. After further washing with PBS, cells were incubated with secondary antibodies for 1 hour. Finally, cells were stained with DAPI (Sigma) for 15 minutes before mounting with FluorSave solution (Calbiochem, San Diego, CA). Anti- SM α-actin antibody (conjugated with Cy3) and anti-β-actin antibody (AC15) were purchased from Sigma (St. Louis, MO). Anti-cytoplasmic-γ-actin isoform (AB3265) antibody was from Millipore (Temecula, CA). Alexa Fluor 488 phalloidin, fluophor 488 donkey anti-sheep, fluophor 555 goat anti-rabbit and mouse antibodies were obtained from Life Technology (Carlsbad, CA). Images were captured with Olympus FV10i LIV confocal microscope (the Cell & Molecular Imaging Shared Resource, Hollings Cancer Center, Medical University of South Carolina) and Zeiss Axio Imager M2 (Molecular Morphology, Medical University of South Carolina). For histologic analysis, liver tissue was fixed in 10% buffered formalin (Fisher, NJ). Picrosirius red staining was performed as before²⁸ and collagen content (area %) was quantitated via image-J.