Rabbit anti mmp 12

Western blots on whole retinas were performed as previously described. 22 Briefly, total protein fractions were separated by SDS-PAGE (5%-20% acrylamide) and immunoblotted after semidry electrotransfer to nitrocellulose membranes (0.2-lm pore). Primary antisera for analyzing the activation and integrity of FAK consisted of rabbit-anti-p-FAK (Tyr397; 1:1000; Cell Signaling Technology, Danvers, MA, USA) and rabbit-anti-FAK (1:1000; Cell Signaling Technology). Primary antisera for evaluating MMP levels in the retina consisted of rabbit-anti-MMP-3 (1:250; Abcam, Cambridge, Cambridgeshire, UK), rabbit-anti-MMP-9 (1:250; Abcam), rabbit-anti-MMP-12 (1:250; Abcam), and rabbit-anti-MMP-2 (1:250; Cell Signaling Technology). Primary antisera were detected with a 1:1000 dilution of secondary antibody (horseradish peroxidase conjugated, cross reacted against rat serum antigens; Jackson Immunoresearch, West Grove, PA, USA). Chemiluminescent immunoreactive complexes were visualized using a Bio-Rad Fluor-S Max imager (Hercules, CA, USA). Loading was verified by visualizing protein bands with Ponceau S dye, and reprobing blots with a rabbit antisera directed against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2000; 2118; Cell Signaling Technology).

Fluorescence immunohistochemistry was performed on transverse retinal sections as previously described. 19, 22, 24, 28 Primary antisera for characterizing MMP localization in the rat retina were rabbit-anti-MMP-2 (1:1000; Cell Signaling Technology, Danvers, MA, USA), rabbit-anti-MMP-3 (1:250; Abcam), rabbit-anti-MMP-9 (1:500; Chemicon, Fisher Scientific, Hampton, NH, USA), and rabbit-anti-MMP-12 (1:500; Abcam), guinea pig anti-RBPMS (RNA binding protein with multiple splicing, 1:1000; Phosphosolutions, Aurora, CO, USA), rabbit anti-GAP-43 (growth associated protein-43, ab16053, 1:1000; Abcam). All primary and secondary antisera were diluted in PBS containing 3% normal donkey serum and 0.3% Triton X-100. Secondary affinity-purified antisera (Cy3 conjugated, Jackson Immunoresearch) were used for fluorescence visualization of MMP immunoreactivity.