At the desired time point, each corneal piece was fixed for 10
minutes in ice-cold methanol and rinsed three times in
phosphate-buffered saline (PBS). Pieces were incubated for 10 minutes
in 1% Triton X-100 in PBS to permeabilize the cells, followed by
incubation for 10 minutes with 4% bovine serum albumin in PBS to block
nonspecific binding. Corneal pieces were then incubated for 2 hours
with mouse monoclonal anti-human Ki67 IgG (clone MIB-1: Zymed
Laboratories, San Francisco, CA) at room temperature in a humidified
chamber. This antibody was prediluted by the supplier and was applied
directly to the corneal pieces. Each piece was then washed three times
with PBS. Fluorescein-conjugated anti-mouse IgG (Jackson Laboratories,
West Grove, PA.), diluted 1:200 in blocking buffer, was applied to the
corneal pieces for 2 hours. Pieces were then washed with PBS and
mounted in medium containing propidium iodide (PI: Vector, Burlingame,
CA) for visualization of nuclei. Slides were viewed using a confocal
microscope (model TCS 4D; Leica, Deerfield, IL) equipped with a laser
(model DMRBE; Leitz Lasertechnik, Heidelberg, Germany) and software
(SCANware ver. 4.2; Leica). Images were collected using a ×16, ×40,
or ×100 oil-immersion lens. Laser power and gain controls were
adjusted to achieve an optimal range of output signal intensity for
each channel. Confocal images were collected, and micrographs were
printed (Photoshop ver. 4.0; Adobe Systems, San Jose, CA). For some
micrographs, the printing contrast was adjusted to provide a clearer
image.