Cellsens standard imaging software version 1

Cultured HIOs were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in PBS, washed in PBS, and embedded in Tissue-Tek O.C.T. (Sakura Finetek, Torrance, CA), then sliced on a cryotome and mounted on Fisherbrand Superfrost Plus slides. The tissue was, then blocked and permeabilized in PBS containing 5% goat serum (Invitrogen, Carlsbad, CA) and 0.5% Triton-X 100 (LabChem, Pittsburgh, PA) for 1 hour at 37 degrees C. An additional blocking step was performed using SFX signal enhancer (Invitrogen, Carlsbad, CA) for 30 minutes at room temperature, followed by incubation with primary antibodies to vimentin (Sigma, St. Louis, MO, catalog # SAB4503083) or to desmin (Abcam, Cambridge, MA, catalog # ab8976) at 1:100 for 2 hours. Slides were washed with 0.05% Tween 20 (Bio-Rad, Hercules, CA) in PBS, followed by incubation with AlexaFluor 488-conjugated anti-mouse or anti-rabbit antibody (Invitrogen, Carlsbad, CA). Detection of αSMA was performed with 30 minute incubation using Cy3 conjugated mouse anti-αSMA (Sigma, St. Louis, MO) at 1:200, co-stained with 4, 6’ diamidino-2-phenylindole (DAPI), (Molecular Probes, Eugene, OR) at 1:1000, to visualize nuclei. Images were acquired using Olympus BX60 microscope and DP72 camera, with CellSens Standard imaging software, version 1.11 (Olympus America, Center Valley, PA). Images were merged using ImageJ software.