After clinical examination, rabbits were humanely killed with an intravenous injection of potassium chloride (1 mg/kg) under deep anesthesia, and corneas were extracted. The half of a cornea was subjected to histologic assays, and another half to molecular assays.
For histologic examination, the frozen sections were subjected to hematoxylin-eosin staining, immunostaining for BrdU, p63, and CK (cytokeratin) 3/12. For detection of BrdU-labelled nuclei, rat mAb to BrdU (1 : 100, ab6326, Abcam) was used. For p63 and CK 3/12 immunostaining, goat mAb to rabbit p63 (1 : 100, ab124762, Abcam), and mouse mAb to rabbit CK3/12 (1 : 100, ab68260, Abcam, Cambridge, UK) were used as primary antibodies. Secondary antibodies used were goat anti-rat IgG, TRITC (Millipore, Billerica Massachusetts 01821, USA) for BrdU and Ck3/12, and goat anti-mouse IgG Alexa 488 (Invitrogen, Waltham, MA, USA) for p63. Nuclei were counterstained using Hoechst 33342 (Sigma, St. Louis, MO, USA).
The stained slides were observed under a fluorescent microscope (BX-61, Olympus, Melville, NY, USA) with ×200 magnification. The number of positively stained cells was counted in three different sections of each eye, and the average count was determined.
For histologic examination, the frozen sections were subjected to hematoxylin-eosin staining, immunostaining for BrdU, p63, and CK (cytokeratin) 3/12. For detection of BrdU-labelled nuclei, rat mAb to BrdU (1 : 100, ab6326, Abcam) was used. For p63 and CK 3/12 immunostaining, goat mAb to rabbit p63 (1 : 100, ab124762, Abcam), and mouse mAb to rabbit CK3/12 (1 : 100, ab68260, Abcam, Cambridge, UK) were used as primary antibodies. Secondary antibodies used were goat anti-rat IgG, TRITC (Millipore, Billerica Massachusetts 01821, USA) for BrdU and Ck3/12, and goat anti-mouse IgG Alexa 488 (Invitrogen, Waltham, MA, USA) for p63. Nuclei were counterstained using Hoechst 33342 (Sigma, St. Louis, MO, USA).
The stained slides were observed under a fluorescent microscope (BX-61, Olympus, Melville, NY, USA) with ×200 magnification. The number of positively stained cells was counted in three different sections of each eye, and the average count was determined.