5-bromo-2'-deoxyuridine (BrDU) labeling and immunohistochemistry

For BrDU labeling of mouse corneal epithelial cells, a dose of 100 mg/kg BrDU in Dulbecco's phosphate-buffered saline (DPBS) was administered (i.p.) to WT and DKO mice. Two hr thereafter, mice were euthanized by carbon dioxide inhalation. The eyes were immediately removed, fixed in 4% paraformaldehyde and embedded in paraffin according to standard procedures. Sagital sections (5 μm) were then deparaffinized, rehydrated and blocked for endogenous peroxidase activity by incubation in 1% H₂O₂. For antigen retrieval, slides were boiled in 10 mM sodium citrate containing 0.05% Tween-20 (pH 6.0) for 15 min and subsequently incubated with pre-activated RNase A (100 μg/ml in 50mM Tris, pH 7.5, Roche, Indianapolis, IN) for 30 min. The RNA-free DNA was then denatured in 4N HCL at room temperature for 5 min. The pH was then neutralized with 50 mM Tris (pH 7.5) and the tissue section was blocked with Tris-NaCl blocking buffer (TNB, 0.10 M Tris.HCl, 0.15 M NaCl, 0.5% blocking reagent (Roche, Indianapolis, IN)) solution in a humidified chamber. For p53 and Ki-67 staining, the RNAse treatment and DNA denaturation steps above were omitted. Primary antibodies (Anti-BrDU antibody 1:100 BD Biosciences, San Jose, CA; Anti-Ki-67, 1:100; Anti-p53 (FL-393) 1:50, SantaCruz, CA) were diluted in TNB, placed on the section and incubated at 4°C overnight. Following washing in DPBS, the tissue section was incubated for 1 hr with horseradish peroxidase (HRP)-conjugated secondary antibody (1:500) after which the signal was amplified by incubation in biotin-tyramide solution (Perkin Elmer, Waltham, MA) for 5 min. Slides were washed and incubated in streptavidin-conjugated HRP for 30 min at RT (25°C). 3-Amino-9-ethylcarbazole (AEC) (BD Biosciences, Bedford, MA) was applied to the section to facilitate visualization of the labeled protein. Immunohistochemical images were captured on a digital camera (Nikon DS-Fi1-L2) fitted to a microscope (Nikon Eclipse E200). Corneal sections from the same animal were hematoxylin and eosin (H&E) stained according to standard procedure.

The corneoscleral junction was used to define the limit of the cornea from limbus to limbus. The mouse cornea was circular and regions from central and peripheral cornea were visualized at 10X magnification. A 1280 X 960 pixel frame was chosen on central and peripheral cornea (200X magnification) of each mouse to count both BrDU-positive cells and the total number of cells from corresponding H&E-stained sections. The number of cells staining positive for BrDU (N_B) was expressed as a percentage of the total number of cells stained by hematoxylin (N_H) as N_B/N_H x 100. Results are presented as mean ± S.E. (N = 3). Total Ki-67 positive nuclei in the entire length of the corneal epithelium of each mouse sagittal eye section were counted at 10X magnification. Results are presented as mean ± S.E. (N = 3).