Conjunctival and scleral tissue specimens were fixed with Carnoy Solution (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) and embedded in paraffin. Next, 5-μm-thick sections were cut, mounted on silanized slides (Dako Japan, Kyoto, Japan), and deparaffinized with xylene and a series of graded ethanol. Then, Azan-Mallory staining for each section was used to identify collagen fibers. The conjunctival and scleral areas of the lesion where the flap was made were assessed by use of a computerized morphometry system (MacSCOPE Ver 2.2; Mitani Corporation, Fukui, Japan), and the ratio of conjunctival area to scleral area was then calculated. The collagen density in the conjunctiva was quantified as follows: the collagen area identified by Azan-Mallory staining was quantified by use of a computerized color extraction system (Win Roof Ver. 6.13; Mitani Corporation), and the collagen density was then calculated as the ratio of collagen area to total conjunctival area.
To retrieve the antigen, the sections were pretreated with 10-mM citrate buffer at pH 6.0, and then autoclaved for 5 minutes at 120°C before immunohistochemical staining. The sections were then soaked in absolute methanol containing 3% hydrogen peroxide for 5 minutes at room temperature to remove endogenous peroxidase activity. Next, the sections were incubated with 10% nonimmune goat serum for 5 minutes to suppress nonspecific binding. For identification of fibroblasts and vessels, anti-bovine vimentin antibody (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and mouse anti-human von Willebrand factor antibody (Wako Pure Chemical Industries, Ltd.), respectively, were used. The sections were incubated overnight at 4°C with each antibody, followed by reaction with appropriate reagents from a streptavidin-biotin peroxidase kit (Dako Denmark A/S, Glostrup, Denmark) and 3-amino-9-ethylcarbazole for 5 to 10 minutes. The sections were then lightly counterstained with hematoxylin. The procedures of immunohistochemical staining with mouse monoclonal antibody against PCNA (PC10) (M0879; Dako Denmark A/S) were as described in our previous report.
20 Fibroblasts, vessels, and PCNA-positive cells were counted at the sites where they accumulated in the conjunctival and scleral lesions by use of a light microscope (number per ×100 field), and the average number of each type of cells or vessels in five randomly selected fields was then calculated.