Gelatin zymography for MMP-9 activity.

Protein was extracted from wounds at day 3 and 7 with Shirmer strips (Alcon; Novartis, Basel, Switzerland). Shirmer strips were placed directly over the wounds and covered for 6 h with a Tegaderm. After 6 h, the strip was removed and protein was eluted from them with a buffer solution made of 50 mM Tris, 50 mM NaCl, 0.05% Brij-35 detergent (Fischer Scientific Waltham, MA) at 4°C for 3 h. We then normalized the protein concentration between the samples by diluting them with sample buffer. The samples were then run at room temperature using a Novex 10% zymogram gel (gelatin) (Invitrogen, Life Technologies), per the manufacturer’s instructions. Murine MMP-9 standard (R&D Systems) was loaded as a control for later quantifying proteolytic activity. Likewise, whole protein was added to later evaluate the ratios of pro-MMP to active MMP. Gels were allowed to renature for 1 h and incubated overnight at 37°C. Band intensity was quantified by using NIH Image J software version 1.49 (https://imagej.nih.gov/ij/).