Masson's trichrome stain for collagen fibers and quantification

Sections were stained with Harris hematoxylin (Newprov, Pinhais, Paraná, Brazil) for 40 s, washed under running tap water for 2 min and stained with Masson's trichome solution for 17 min. Images of consecutive regions of the wound area were taken in × 200 magnification using an optic microscope (Axioskop 40 Zeiss; Carl Zeiss). The identification of collagen fibers was made by deconvolution of color images on ImageJ software Version 1.52 (NIH, Bethesda, Maryland, USA). During color image deconvolution, images with Masson’s trichrome stain were deconvolved, and the green component was identified as the collagen fibers. The area of the green collagen fibers was measured using the “threshold” tool. The threshold was manually adjusted until the entire green area was highlighted in red. Then, the threshold was measured as the percentage of the stained area⁵⁸.