Human TSP-1 cDNA, corresponding to base pairs 3874-4375 of the previously reported human TSP-1 cDNA (GenBank accession number NM_003246; http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD), was subcloned into the pGEM-T vector (Promega, Madison, WI) and used for the generation of sense and antisense RNA probes. Digoxigenin (DIG)-labeled RNA probes were prepared with DIG RNA labeling mix (Roche, Basel, Switzerland).
Fresh human corneal-, limbal-, and conjunctival epithelia obtained from the eye bank were dissected, fixed, and embedded in paraffin by using proprietary procedures, and 6-μm sections were cut (n = 3; eyes aged 36.7 ± 16.5 years). They were deparaffinized with xylene, rehydrated with an ethanol series and phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde in PBS (15 minutes), and then washed with PBS. Then the sections were treated with 10 μg/mL proteinase K in PBS (30 minutes, 37°C), washed with PBS, refixed with 4% paraformaldehyde in PBS, washed again with PBS, and placed in 0.2 M HCl for 10 minutes. After they were washed with PBS, the sections were acetylated by a 10-minute incubation in 0.1 M triethanolamine-HCl (pH 8.0) and 0.25% acetic anhydride. After they were again washed with PBS, they were dehydrated through an ethanol series. After 55°C, 16-hour hybridization to probes (100 ng/mL) in probe diluent (Genostaff, Tokyo, Japan), the sections were washed in 5× hybridization buffer equal to 5× SSC (HybriWash; Genostaff) at 55°C for 20 minutes and then in 50% formamide, 2× hybridization buffer (55°C, 20 minutes; HybriWash, Genostaff). This was followed by RNase treatment in 50 μg/mL RNase A in 10 mM Tris-HCl (pH 8.0), 1 M NaCl, and 1 mM EDTA. The sections were washed twice with 2× hybridization buffer (55°C, 20 minutes), twice with 0.2× of the buffer (55°C, 20 minutes), and once with TBST (Tris-buffered saline-0.1% Tween-20). After 30-minute treatment with 0.5% blocking reagent (Roche) in TBST, the sections were incubated for 2 hours with anti-DIG AP conjugate (Roche) diluted 1:1000 with TBST. They were then washed twice with TBST and incubated in 100 mM NaCl, 50 mM MgCl2, 0.1% Tween-20, and 100 mM Tris-HCl (pH 9.5). After an overnight color reaction with the BM purple alkaline phosphatase (AP) substrate (Roche), the sections were washed with PBS, dehydrated, and mounted on coverslipped glass slides (Malinol; Mutoh Chemical Co., Tokyo, Japan).