Gelatinase Zymography of Pericyte Secretome

Gelatinase zymography was performed as previously described with modifications [18 (link)]. Briefly, isolated microvessels were homogenized in lysis buffer, and the pericytes conditioning media (1.5 ml) was concentrated to 20–30 μl using microcon spin columns (Millipore, Bedford, MA). The samples were separated on an 8% polyacrylamide SDS gel containing 1 mg/ml gelatin. The gel was then soaked in zymogram renaturing buffer (Novex, Shanghai, China) for 30 min at room temperature and incubated for 16 h at 37 °C in zymogram developing buffer (Novex, Shanghai, China). To visualize the MMPs, the gel was stained with SimplyBlue Safe Stain (Invitrogen, Shanghai, China) for 30 min. The gel was imaged using a CCD camera and analyzed using ImageJ software (NIH, Bethesda, MD, USA).