Eyes from mice 1 day p.i. were embedded in OCT compound (Sakura Finetek, Torrance, CA) and snap-frozen in liquid nitrogen. Frozen tissues were sectioned on a cryostat at 15-μm thickness and processed with Gill hematoxylin and eosin or periodic acid-Schiff (PAS) stains (Sigma-Aldrich, St. Louis, MO).
For immunofluorescent staining frozen corneal sections were thawed, dehydrated, and fixed in 2% paraformaldehyde at 4°C for 10 minutes. Sections were blocked with 10% normal donkey serum (Jackson ImmunoResearch Laboratories, Philadelphia, PA) in PBS for 1 hour to decrease nonspecific binding. The following primary antibodies were diluted 1:100, applied to the blocked sections, and incubated overnight at 4°C: polyclonal rabbit antibody MMP-2 (29575, AnaSpec, San Jose, CA), MMP-8 (29578, AnaSpec), MMP-9 (AB19016, Millipore, Billerica, MA), and TIMP-1 (sc-5538, Santa Cruz Biotechnology, Santa Cruz, CA), or goat antibody MMP-13 (sc-12363, Santa Cruz Biotechnology). Secondary Alexa-Fluor 488-conjugated donkey anti-rabbit or anti-goat (Invitrogen, Carlsbad, CA) antibodies were then applied and incubated in a dark chamber for 1 hour at room temperature followed by washing and counterstaining with propidium iodine (Invitrogen) in gel (Gel/Mount; Biomeda, Foster City, CA), and a coverslip was applied. Sections were observed with a laser-scanning confocal microscope (LSM 510 with krypton-argon and He-Ne laser; Zeiss, Thornwood, NY) with 488- and 543-nm excitation and emission filters (LP 505 and LP 560). Images were acquired with a 40× oil-immersion objective and processed using Zeiss LSM-PC software.
Immunohistochemistry was performed using a similar protocol to immunofluorescent staining. After fixation with 2% paraformaldehyde, corneal sections were serially treated with 0.3% hydrogen peroxide and 10% normal donkey serum. Primary antibodies were applied (MMP-2, 1:200; MMP-8, 1:200; MMP-9, 1:200; MMP-13, 1:100; and TIMP-1, 1:200). After incubation, biotin-conjugated donkey anti-rabbit or anti-goat (Jackson ImmunoResearch Laboratories, West Grove, PA) secondary antibodies were applied and incubated, followed by reagent (Vectastain Elite ABC Kit, Vector Laboratories, Burlingame, CA). The samples were incubated with diaminobenzidine (DAB) as a chromogen (Vector Laboratories) followed by counterstaining with hematoxylin. Sections were dehydrated, coverslipped, and photographed with an epifluorescent microscope.