Hematoxylin and eosin

Skin tissue was immersed in Tissue-Tek O.C.T. Compound (Sakura), frozen, and 5 μm sequential sections were cut using a MICROM HM525 cryomicrotome (Thermo Fisher Scientific). Only sections from the center of the wound were used for analysis. Slides were fixed in 2% paraformaldehyde and blocked in 5% normal goat serum (Sigma) for 1 hour at 37 °C. Primary antibodies used included rabbit antiwide spectrum cytokeratin (1: 200, Abcam), rabbit anti-Ki-67 (1: 400, Abcam), rabbit antivon Willebrand Factor (Vwf) (1: 400, Abcam), and anti-alpha smooth muscle actin (α-SMA) (1: 500, Sigma). Primary antibodies were incubated for either 1 hour at 37 °C or overnight at 4 °C. Goat anti-rabbit IgG Alexa Fluor 488 and Alexa Fluor 594-conjugated secondary antibodies (1: 200, Invitrogen) were incubated for 45 m at 37 °C. All slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma). Slides were also stained with hematoxylin and eosin (Ricca) or with Picosirius Red Stain Kit (Polysciences Inc.) following the manufacturer’s protocols. All images were acquired using an Eclipse Ti inverted microscope (Nikon) and all image analyses were performed using NIS Elements Version 3.2 software (Nikon).