Groups of rabbits were killed on day 1 (n = 4), day 7
(n = 4), day 14 (n = 5), and day 28 (n= 6) for histologic examination. Intact eyes including eyelids
were removed en bloc by orbital exenteration and fixed by immersion in
4% formaldehyde overnight. The portion of each eye that included
either the grafted or ungrafted wound site, including the underlying
scleral bed, was dissected and embedded in paraffin. Sections were cut
on a microtome at 7 μm and stained with H&E for general cell
morphology and Masson’s trichrome to assess collagen deposition and
remodeling (reorganizing). Identification of specific cell types was
based on cell morphology observed in sections stained with H&E.
Polymorphonuclear neutrophils (PMNs) were identified by characteristic
multilobed nuclei and fine cytoplasmic granules. Eosinophils were
characterized by bilobed or multilobed nuclei and larger eosinophilic
granules. Basophils possessed less lobulated, pale nuclei with
cytoplasmic granules that were less numerous and variable in size.
Fibroblasts were characterized by elongated morphology and oval nuclei.
Slides processed for histology were also viewed under cross-polarizing
filters to visualize the organization and alignment of collagen fibers.
Additional tissue sections were stained with an antibody for α-SMA to
identify cells with contractile potential (myofibroblasts). Briefly,
sections were deparaffinized, rehydrated, washed three times (5 minutes
each) with PBS, and blocked with 10% normal goat serum (Gibco,
Gaithersburg, MD). A monoclonal antibody for α-SMA (Sigma, St. Louis,
MO) was used at a dilution of 1:400. To verify the specificity of the
primary antibody, adjacent tissue sections were similarly processed
except for the replacement of the primary antibody with nonimmune mouse
serum. Slides were then incubated with biotinylated goat anti-mouse IgG
(Sigma) diluted 1:400, reacted with avidin-biotin complex (Vector,
Burlingame, CA), and developed with AEC chromogen (Sigma), which yields
a red-brown color indicating positive stain.