All slides were processed for immunohistochemical staining and examined by light microscopy. The antibodies used were vWF and thrombomodulin, markers for the platelet coagulation system; CD34, a marker of blood vessels; PECAM-1 and E-selectin, markers for endothelial cells that are used to determine whether ECSCs are involved in inflammation similar to the endothelial cells of blood vessels; and D2-40, a marker for lymphatic vessels.
Paraffin sections of 2-μm thickness, adjacent to sections stained with hematoxylin-eosin, were mounted on silanized slides and dried overnight at 37°C followed by 1 hour at 60°C. The sections were deparaffinized in xylene and rehydrated in a graded series of alcohol solutions. The slides were pretreated with 0.1% trypsin for vWF and thrombomodulin for 30 minutes; at 121°C for PECAM-1, E-selectin, and D2-40 for 15 minutes; and with a fuschin substrate (Histofine kit [pH 9.0]; NichireiBioscience, Tokyo, Japan) for 15 minutes and cooled for 15 minutes. Immunostaining was performed with an autostainer (Ventana Medical System, Inc. Tucson, AZ).
After the slides were washes with phosphate-buffered saline (PBS; pH 7.2) and in Tween 20, endogenous peroxidase activity was neutralized by 1% H2O2 in methanol for 20 minutes and washed with PBS. The sections were incubated with antibodies for 60 minutes at the following dilutions: vWF, clone F8/86, dilution 1:50 (DAKOCytomation, Kyoto); thrombomodulin, dilution 1:50 (DAKOCytomation); CD34, clone QBEnd10, dilution 1:1 (Immunotech, Westbrook ME); PECAM-1, clone JC70A, dilution 1:20 (DAKOCytomation); E-selectin, dilution 1:1000 (R&D Systems, Minneapolis, MN); and D2-40, clone D2-40, monoclonal dilution 1:1 (Covance, Madison, WI). The sections for E-selectin were incubated with a paraffin section anti-goat immunostain (MAX-PO; NichireiBioscience) as a second antibody and those for the other antibodies were incubated with a multi-antibody immunostain (MAX-PO; NichireiBioscience). The immnocomplex was made visible by 150 mL of 0.05 M Tris HCl buffer solution with 30 μL of H2O2 and DAB for 30 minutes and washed with PBS. The sections were counterstained with hematoxylin for 1 minute, dehydrated, and mounted. These slides were examined and graded by one ophthalmologist and one pathologist who were masked to the source of the slides.