All the procedures followed in this study accorded with the Association for Research in Vision and Ophthalmology Statement for Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee of National Taiwan University Hospital.

1. Isolation, Primary Culture Preparation, and Immunostaining of Bovine CECs

1. Acquire fresh bovine eyes from a local abattoir.
2. Disinfect the eyes in a 10% w/v povidone-iodine solution for 3 min. Wash them with phosphate-buffered saline (PBS) solution.
3. Harvest the corneal button with a scalpel and scissors under sterile conditions. Peel the Descemet's membrane with forceps under a dissecting microscope (note that in this study, the bovine eyes were enucleated by a local abattoir; therefore, the first study procedure was the disinfection of the preenucleated eyes in the laboratory).
4. Incubate the Descemet's membrane in 1 ml of trypsin at 37 °C for 30 min. Collect the bovine CECs by centrifugation at 112 x g for 5 min. Resuspend the cells in 1 ml of supplemented hormonal epithelial medium (SHEM) containing equal volumes of HEPES-buffered Dulbecco's modified Eagle medium and Ham's F12 medium, supplemented with 5% fetal bovine serum, 0.5% dimethyl sulfoxide, 2 ng/ml of human epidermal growth factor, 5 mg/ml of insulin, 5 mg/ml of transferrin, 5 ng/ml of selenium, 1 nmol/l of cholera toxin, 50 mg/ml of gentamicin, and 1.25 mg/ml of amphotericin B.
5. Seed the cells (approximately 1 x 10⁵ cells per eye) into a 6 cm dish. Culture them in the SHEM. Incubate the dish at 37 °C in an atmosphere of 5% CO₂ in air. Change the culture medium every 3 days.
6. When the cells reach confluence, wash them in PBS and incubate them in 1 ml of trypsin at 37 °C for 5 min. Collect them by centrifugation at 112 x g for 5 min. Re-suspend the cell pellet in 1 ml of the SHEM.
   1. Count the cells in a hemocytometer. Seed the cells on cover slides at a density of 1 x 10⁴ per well in a 24-well plate, and culture the cells in the SHEM. For investigating the EnMT-suppressing effect of marimastat, incubate the cells in SHEM with 10 µM of marimastat added into the culture medium, and change the culture medium every 3 days.
7. Fix the cells at an indicated time point with 250 µl of 4% paraformaldehyde, pH 7.4, for 30 min at room temperature. Permeabilize with 250 µl of 0.5% Triton X-100 for 5 min. Block with 10% bovine serum albumin for 30 min.
8. Incubate the cells with primary antibodies against active beta-catenin (ABC), snail, and slug overnight at 4 °C. The dilution of the primary antibodies used is 1:200. The antibodies are diluted in antibody diluent composed of 10 mM PBS, 1% w/v bovine serum albumin, and 0.09% w/v sodium azide.
9. Wash the cells twice with PBS for 15 min, and incubate them with the Alexa Fluor-conjugated secondary antibody (1:100 in antibody diluent) at room temperature for 1 hr.
10. Counterstain the cell nucleus by covering the cells with 2 µg/ml of 4',6-diamidino-2-phenylindole, wash the cells twice with PBS for 15 min, and mount them with anti-fading mounting solution.
11. Obtain immunofluorescent images at the excitation wavelengths of 405 and 488 nm by using a laser scanning confocal microscope with 20X objective magnification.

2. Rat Corneal Endothelium Cryoinjury Model and Intracameral Injection

1. Anesthetize 12 week-old male Sprague-Dawley rats with intramuscular injections of 2% xylazine (5.6 mg/kg) and tiletamine plus zolazepam (18 mg/kg). Gently pinch the skin of the animals to confirm proper anesthesia in the absence of skin twitching.
2. Instill one drop of 0.5% proparacaine hydrochloride to the right eye of each rat to minimize eye pain and the blink reflex. Instill tetracycline ointment to the left eye to prevent corneal dryness.
3. Cool a stainless steel probe (diameter = 3 mm) in liquid nitrogen. Apply the stainless steel probe to the central cornea of the right eye for 30 sec. Frequently instill PBS to the right eye during the procedure to prevent corneal dryness.
4. Instill 0.1% atropine and 0.3% gentamicin sulfate immediately after cryoinjury and once daily to relieve ocular pain resulting from ciliary spasm and to prevent infection.
   1. After the procedure, keep the rats warm using a heat lamp, and observe their recovery every 15 min after anesthesia until they regain motor control. Additionally, apply tetracycline ointment to the right eye to prevent corneal dryness during the recovery period.
5. Repeat the corneal cryoinjury for 3 consecutive days.
6. For delivering marimastat or bFGF into the anterior chamber of the rat eyes, anesthetize the rats as described previously. Instill one drop of 0.5% proparacaine hydrochloride into the right eye to minimize eye pain and the blink reflex.
7. Irrigate the ocular surface with sterile PBS. Perform anterior chamber paracentesis under an operating microscope by inserting a 30 G needle attached to a 1 ml syringe at the paralimbal clear cornea in a plane above and parallel to the iris.
8. Turn the needle bevel up, and slightly depress the corneal wound to drain some aqueous humor and reduce the intraocular pressure. Inject 0.02 ml of the drug intracamerally. Gently compress the needle tract with a cotton tip during the needle withdrawal.
9. Photograph the external eye at an indicated time point under the operating microscope.

3. Harvesting the Rat Corneal Button and Immunostaining

1. To euthanize the rats, place them in a euthanasia chamber and infuse 100% CO₂ at a fill rate of 10-30% of the chamber volume per minute. Maintain CO₂ infusion for an additional minute after lack of respiration and faded eye color.
2. Penetrate the rat eye at the limbus with a sharp blade. Cut the corneas with corneal scissors along the limbus. Flatten the corneas on a slide. Make additional radial incisions if the corneas curl up.
3. Fix the corneas with 250 µl of 4% paraformaldehyde, pH 7.4, for 30 min at room temperature. Permeabilize with 250 µl of 0.5% Triton X-100 for 5 min, and block with 10% bovine serum albumin for 30 min.
4. Incubate the corneas with primary antibodies against ABC overnight at 4 °C with a dilution of 1:200 in antibody diluent. Wash the corneas twice with PBS for 15 min, and incubate them with the secondary antibody (1:100 in antibody diluent) at room temperature for 1 hr. Counterstain the cell nucleus by covering the cells with 2 µg/ml of 4',6-diamidino-2-phenylindole, and wash the cells twice with PBS for 15 min.
5. Mount the corneas in anti-fading mounting solution. Obtain the immunofluorescent images at excitation wavelengths of 405 and 561 nm with a laser scanning confocal microscope at 20X objective magnification.