Quantification of Angiogenesis in Wound Healing

Healed wounds were harvested, bisected, and fixed in 4% paraformaldehyde for 12 hours at 4°C. Fixed tissue was dehydrated and embedded in paraffin blocks. 8-μm-thick sections were serially cut and incubated with a polyclonal rabbit anti-mouse anti-CD31 primary antibody (1:100, Abcam, Cambridge, UK) overnight at 4°C, followed by Alexa Fluor 594 Goat Anti-Rabbit IgG secondary at room temperature (1:200, Invitrogen) for one hour. All samples were counterstained with DAPI. Slides were mounted with the Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA) and cover-slipped. A Zeiss Axioplan 2 fluorescence microscope was used to image the slides (Carl Zeiss, Inc., Thornwood, NY). Quantification of fluorescence was performed by a blinded observer analyzing at least five high-powered fields per wound at 200x using ImageJ software (NIH) as previously described [17 (link)].