Vectashield with 4 6 diamidino 2 phenylindole

Corneas were excised on day 14 after suture-induced CNV and washed with phosphate-buffered saline. The corneal epithelium was removed after incubation with 20 mM EDTA for 60 min at 37 °C, fixed in acetone for 15 min at 20–22 °C, and blocked in 2% bovine serum albumin for 60 min. Corneas were then double-stained for CD31 and lymphatic vessel endothelial hyaluronan receptor LYVE-1 as described previously by using overnight incubation with goat anti-mouse CD31 FITC (1:100; Santa Cruz Biotechnology, Dallas, TX, USA) and goat anti-mouse LYVE-1 (1:400; AF2125, R&D Systems, MN, USA)^{41 (link),54 (link)}. A Cy3-conjugated donkey anti-goat antibody (1:2,000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was then added as the secondary antibody and incubated for another 2 h. Stained whole mount corneas were mounted in Vectashield with 4,6-diamidino-2-phenylindole (Vector Laboratories Inc., Burlingame, CA, USA). Images of stained whole mount corneas were taken under a fluorescence microscope (BZ-X71000, KEYENCE, Osaka, Japan). The fractions of total corneal areas covered by blood and lymphatic vessels were then calculated by using ImageJ (National Institutes of Health, Bethesda, MD, USA; available at http://rsb.info.nih.gov/ij/index.html)^{41 (link),55 (link)}.

The ovaries were dissected in phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde in PBS for 20 min at room temperature and washed three times in PBS with 0.5% Triton X-100 (PBT) for 30 min. Then, the tissues were mounted in 80% VECTASHIELD with 4′,6-diamidino-2-phenylindole (Vector Laboratories Inc.).