Neonatal (1–14 days of age), juvenile (15 days to 6 weeks of
age), and adult (3–4 months of age) rats were killed. Eyes with or
without eyelids were enucleated, placed in optimal cutting temperature
compound (Tissue Tek II; Laboratory Tek, Napierville, IL) and frozen on
dry ice. Six-micrometer cryostat sections were placed on gelatin-coated
slides and air dried overnight at 37°C. Slides were fixed in 100%
methanol for 10 minutes at −20°C, washed with phosphate-buffered
saline (PBS) for 10 minutes, and then blocked in 1% bovine serum
albumin solution in PBS for 10 minutes. Antibody against pRb was
obtained from Boehringer Mannheim (Indianapolis, IN), antibodies
against p107 (C-18), p130 (C-20), E2F-1 (KH95), E2F-2 (L-20), and E2F-4
(C-20) were obtained from Santa Cruz Biotech (Santa Cruz, CA), and
anti-Ki67 (MM1) from Novacastra (Newcastle-upon-Tyne, UK). The
following dilutions were used for primary antibodies: 2 μg/ml pRb,
E2F-2, E2F-4, and Ki67; 1 μg/ml p107 and p130; and 4 μg/ml E2F-1.
Slides were incubated for 1 hour in a moist chamber at room temperature
for all antibodies except pRb and E2F-1, which were incubated overnight
at 4°C. Slides were rinsed with PBS and then blocked with 1% bovine
serum albumin for 10 minutes. Fluorescein isothiocyanate–conjugated
affinity-purified donkey anti-rabbit IgG (for p107, p130, E2F-2, and
E2F-4) and anti-mouse IgG (for pRb, Ki67, and E2F-1; Jackson
ImmunoResearch, West Grove, PA) were applied at a dilution of 1:50 and
incubated for 1 hour at room temperature. Slides were rinsed in PBS,
and coverslips were mounted with Vectashield (Vector, Burlingame, CA).
Sections were viewed and photographed with a microscope
(Axiophot; Carl Zeiss, Thornwood, NY). Negative controls
consisted of secondary antibody alone, irrelevant monoclonal
antibodies, or primary antibody preadsorbed with its respective
antigen. Sections from at least four rats were examined for each
antibody and time point. Antibody binding was examined in the limbal
and corneal epithelia and in stromal keratocytes. In addition, staining
patterns were examined to determine whether localization was
cytoplasmic, nuclear, or both. Relative percentages of Ki67 binding
were determined by comparing Ki67-labeled cells with total cells by
means of by propidium iodide counterstaining. Cells were counted in the
basal cell layers of limbal and corneal epithelia and in the corneal
stroma. Ki67-labeled keratocytes were not quantitated in the limbal
stroma because this area contains a mixture of cell types.