Wound Closure and Collagen Quantification

Wounds were photographed with a camera (Canon PowerShot A640), and the images were analyzed by calculating the enclosed pixel area with the NIH Image J software. The percentage of wound closure was calculated by using the following equation: Wound closure (%) = (Wound area on day 0 –Wound area on the indicated day) / Wound area on day 0 x 100% [14 (link)]. Collagen contents in paraffin-embedded skin sections were determined by using quantitative micro-assay kit (Chondrex, Inc, Redmond, WA) following the manufacturer’s instructions [15 (link)]. This method is based on the selective binding of Sirius Red and Fast Green to collagens and non-collagen proteins. Briefly, 10 mm-thick sections were deparaffinized, and stained with Sirius Red and Fast Green. The color in the tissue sections was eluted by dye extraction solution. Absorbance was measured in a spectrophotometer at OD540 (for Sirius Red) and OD605 (for Fast Green), respectively. The relative collagen amount is calculated as the ratio of OD values of collagens to non-collagen proteins.