Immunohistochemical and Immunofluorescence Analysis

Fixed and paraffin tissue sections were processed as described before [48 (link)]. Primary antibodies, α-SMA (ab5694) and FSP1 (ab27957) from Abcam (Cambridge, MA, USA), Ki67 (94495) from Cell Signaling, (Danvers, MA, USA), Vimentin (SC7557) from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), and Pan-cytokeratin (F0397) from (MilliporeSigma, St. Louis, MO) were used. For IHC, secondary antibody application was followed by incubation with Streptavidin HRP conjugate (Invitrogen, Carlsbad, CA, USA), and DAB (Vector Laboratories, Burlingame, CA, USA), and counterstained with Harris Haematoxylin, dehydrated, and mounted with Permount (Fisher Scientific, Waltham, MA, USA). For IF, goat anti-Rabbit IgG fluor and Goat anti-mouse IgG Texas red (Invitrogen, Carlsbad, CA, USA), secondary antibodies were applied, incubated, washed and stained with DAPI, and mounted with Flour-G (Invitrogen, Carlsbad, CA, USA). Images were captured using a Nikon 80i upright microscope (Tokyo, Japan).