Anti ki67

Immunohistochemistry (IHC) was performed on 7‐, 10‐, or 20‐μm‐thick frozen skin sections fixed in 2% paraformaldehyde or ice‐cold acetone, using the following primary antibodies: anti‐pan‐keratin antibodies were as previously described (Rittié et al., 2013); anti‐Ki67 was from Biogenex (Fremont, CA, USA); anti‐β1‐integrin was from EMD Millipore (Temecula, CA, USA); and anti‐laminin‐γ2 was from AbCam (Cambridge, MA, USA). IHC was performed as previously described (Rittié et al., 2008), and slides were mounted with 90% glycerin or ProLong Gold medium (both from Thermo Fisher Scientific, Waltham, MA, USA). Imaging of horizontally cut sections was performed with a Leica MXFL III Stereo Microscope (Leica Microsystems, Buffalo Grove, IL, USA; accessed at the Microscopy and Image‐Analysis Laboratory, Biomedical Research Core Facilities, University of Michigan Medical School). Other imaging was performed with an Axioscope 2 (Carl Zeiss, Thornwood, NY, USA) equipped with a Spot digital camera (Spot Imaging Solutions, Sterling Heights, MI, USA). Large areas were captured on multiple overlapping sections, and images were merged using Photostitch 3.1 software (Canon, Lake Success, NY, USA). A microscope micrometer (Thermo Fisher Scientific) was used during imaging for image calibration.

Antibodies used in this study were as follows: anti-CCN1 (Cat# 14479S, Cell Signaling Technology, Danvers, MA, USA, for Western Blots, Cat# HPA029853, Sigma-Aldrich, St. Louis, MO, USA, for immunostaining), anti-Keratin10 (K10) (Cat# ab76318, Abcam, Cambridge, UK), anti-Ki67 (Cat# MU297-UC, Biogenex, Fremont, CA, USA), anti-integrin-β1 (Cat# MAB1965, Sigma-Aldrich), anti-Yes-Associated Protein (YAP) (Cat# sc-101199, Santa Cruz, Dallas, TX, USA), anti-type7 collagen (COL7) (Cat# ab93350, Abcam), anti-elastin (Cat# MAB2503, Abnova, Taipei, Taiwan), anti-β-actin (Cat# sc-47778, Santa Cruz), goat anti-rabbit IgG-HRP (Cat# A16104, Thermo Fisher Scientific, Waltham, MA, USA) and goat anti-mouse IgG-HRP (Cat# G-21040, Thermo Fisher Scientific). Alexa Fluor conjugated secondary antibodies were used for the visualization of immunostaining (Thermo Fisher Scientific). 4% Paraformaldehyde Phosphate Buffer Solution (4% PFA) (Cat# 161-20141) and plasmin solution from human plasma (Cat# 166-24231) were purchased from FUJIFILM Wako Pure Chemical Corp. (Osaka, Japan). The THG used in this study was a kind gift of NAGASE & CO., LTD (Tokyo, Japan).