Alexa fluor 594 conjugated griffonia simplicifolia isolectin b4

Anesthetized mice were perfused through the left ventricle with PBS followed by 5 mg/ml fluorescein isothiocyanate (FITC)-labeled dextran (MW=10,000 Da; Sigma, St. Louis, MO). The eyes were enucleated and fixed in a 4% paraformaldehyde solution. The anterior segment and retina were removed from the eyecup, and the remaining RPE/choroid/sclera complexes were flatmounted after four radial incisions. The dissected RPE/choroid/sclera were treated with blocking solution (5% BSA, 5% normal donkey serum, and 0.5% Triton X-100 in PBS) for 1 h at room temperature and incubated with Alexa Fluor^® 594-conjugated Griffonia simplicifolia isolectin B4 (1:100 dilution; Invitrogen) overnight at 4 °C. The samples were washed three times with PBS and mounted sclera side down on microscope slides. Images of CNV lesions were obtained using a fluorescence microscope (Olympus), and the CNV area in each image was calculated using ImageJ software (NIH). For the analysis of CNV volumes, Z-stack images of CNV lesions were acquired with an LSM780 confocal microscope (Zeiss, Jena, Germany). The sum of the CNV area in each Z-stack layer (multiplied by 5-μm thickness) was used as the volume of the CNV lesion [11 (link)].

The eyes were enucleated, fixed, embedded in optimum cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA) or paraffin, sectioned, and mounted on slides. First, the endogenous peroxidase activity in the tissues was quenched. The sections were blocked with 10% normal goat serum and were then incubated with primary immunoglobulin G (IgG) against phospho-Y416 Src (p-Y416 Src; Invitrogen) or glial fibrillary acidic protein (GFAP; Dako), followed by the appropriate fluorescent secondary IgG (Dako). Blood vasculature was visualized by costaining with Alexa Fluor^® 594-conjugated Griffonia simplicifolia isolectin B4 (Invitrogen). Cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA). The images were acquired using a fluorescence microscope (Olympus), and six to eight sections were examined per group.

The eyes were enucleated and fixed overnight in 4% paraformaldehyde. The retinas were isolated, cut four times from the edge to the center, soaked in blocking solution (5% BSA [BSA], 5% normal donkey serum, and 0.5% Triton X-100 in PBS), and then incubated overnight at 4 °C with Alexa Fluor^® 594-conjugated Griffonia simplicifolia isolectin B4 (1:100 dilution; Invitrogen, Carlsbad, CA) in PBS containing 1 mM CaCl₂. Retinas were rinsed three times with PBS, flatmounted on microscope slides, and embedded in fluorescent mounting medium (Dako, Carpinteria, CA). Images of the whole-mounted retina were captured using a fluorescence microscope (Olympus, Tokyo, Japan); the exposure and gain were kept constant for all samples. In each image, the number of pixels in the preretinal neovascular tufts was determined using ImageJ software (National Institutes of Health, Bethesda, MD) and was expressed as a percentage of the number of pixels in the entire retinal area.