Two weeks after laser treatment, the sizes of CNV lesions were measured in choroidal flatmounts.
18 Mice used for the flatmount technique were anesthetized and perfused with 1 mL phosphate-buffered saline (PBS) containing 50 mg/mL fluorescein-labeled dextran (2 × 10
6 average molecular weight; Sigma, St. Louis, MO), as previously described.
19 The eyes were removed and fixed for 1 hour in 10% phosphate-buffered formalin. The cornea and lens were removed, and the entire retina was carefully dissected from the eyecup. Radial cuts (four to seven; average, five) were made from the edge to the equator, and the eyecup was flatmounted in aqueous medium (Aquamount; BDH, Poole, UK) with the sclera facing down. Flatmounts were examined by fluorescence microscopy (Axioskop; Zeiss, Thornwood, NY), and images were digitized using a three-color charge-coupled (CCD) video camera (IK-TU40A; Toshiba, Tokyo, Japan) and a frame grabber. Image analysis software (Image-Pro Plus; Media Cybernetics, Silver Spring, MD) was used to measure the total area of hyperfluorescence associated with each burn, corresponding to the total fibrovascular scar. The areas within each eye were averaged to obtain one experimental value, and mean values were calculated for each treatment group and compared by Student’s unpaired
t-test.
Some mice were killed 2 weeks after laser treatment, and eyes were rapidly removed and frozen in optimum cutting temperature embedding compound (OCT; Miles Diagnostics, Elkhart, IN). Frozen serial sections (10 μm) were cut through the entire extent of each burn and histochemically stained with biotinylated G. simplicifolia lectin B4 (GSA; Vector Laboratories, Burlingame, CA), which selectively binds vascular cells. Slides were incubated in methanol-H2O2 for 10 minutes at 4°C, washed with 0.05 M Tris-buffered saline, pH 7.6 (TBS), and incubated for 30 minutes in 10% normal porcine serum. Slides were incubated 2 hours at room temperature with biotinylated GSA, and after rinsing with 0.05 M TBS, they were incubated with avidin coupled to peroxidase (Vector Laboratories) for 45 minutes at room temperature. After a 10-minute wash in 0.05 M TBS, slides were incubated with HistoMark Red (Kirkegaard & Perry, Cabin John, MD), to give a red reaction product that is distinguishable from melanin, and counterstained with Contrast Blue (Kirkegaard & Perry).