Dm5500b if microscope

Livers were fixed in 4% paraformaldehyde (Polysciences, Inc., Warrington, PA) and embedded in paraffin or Tissue-Tek® Optimum Cutting Temperature (OCT) Sakura Finetek, Torrance, CA) for sectioning. Immunofluorescence (IF) staining signals for PROM1 (1:50 dilution, eBiosciences, San Diego, CA), CYTOKERATIN-19 (1:100 dilution, gift from JR Friedman, University of Pennsylvania, Philadelphia. PA), and LacZ (1:400, Bioss, Woburn, MA) were detected by secondary antibodies conjugated either with anti-mouse cyanine (Cy) 3, Cy5, anti-rat Cy3/Cy5, or anti-rabbit Cy3/Cy5 (1:200 dilution, Jackson ImmunoResearch Labs, West Grove, PA) (9 (link), 10 (link), 20 (link)). Images were then acquired by a Leica DM5500B IF microscope using Leica Suite Advanced Fluorescence (LAS AF) 6000 software (Leica Microsystems, Wetzlar, Germany). Immunohistochemistry was performed using 2.1 μg/mL anti-INTEGRIN-β6 (gift from Shelia Violette, Biogen, Inc). Bright-field images were captured using a Leica DM1000 (DFC290) transmitted light microscope (Leica Microsystems AG, Heerbrugg, Switzerland) with Leica Application Suite (version 2.7.1R1).