Fitc conjugated concanavalin a

One week after laser injury, mice were perfused transcardially with PBS and then with FITC-conjugated concanavalin A (20 μg/mL in PBS; Vector Laboratories, Burlingame, CA) to label the CNV lesions. The eyes were harvested and the RPE/choroids prepared as flatmounts. CNV lesions in the choroidal flatmounts were observed using a fluorescence microscope (Keyence Corporation, Tokyo, Japan). Lesion area was measured by using Image J software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsbweb.nih.gov/ij/index.html) in a masked manner. The outline of the CNV was drawn around the perimeter of the lesion and then the total lesion area was measured. Mean CNV area obtained from the 4 lesions in each eye was used to give a single value for analysis. Each treatment group consisted of 6 or 8 mice.

The retinal vascular leukostasis assay was performed as described previously^{38 (link)}. Briefly, rats were deeply anesthetized, and PBS was injected into the left ventricle. The anesthetized rats were perfused with PBS to remove nonadherent leukocytes in vessels. After injection of PBS, FITC-conjugated concanavalin-A (40 μg/ml) (Vector Laboratories, Burlingame, CA, USA) was injected into the left ventricle. The retinas were surgically isolated and then flat mounted. FITC-labeled adherent leukocytes in the vasculature were counted under a fluorescence microscope by an operator masked to treatment allocation.

Adult C57BL/6 mice received an intravitreal injection of 1 μL containing 1 μg of AXT107 in one eye or 1 μL of PBS in the fellow eye. Twenty-four hours later, both eyes were treated with 50 ng of recombinant mouse TNFα (BioLegend, San Diego, CA, USA) by intravitreal injections. After 24 h, mice were anesthetized with a mixture of ketamine and xylazine hydrochloride and then divided in two groups: (1) Vitreous samples from both eyes were collected to measure albumin levels using the albumin ELISA kit (ab108791, Abcam, Cambridge, MA, USA); (2) the total number of leukocytes adhering to the retinal vessels was counted. For the second group of animals, mice were first perfused with 5 mL of PBS to wash out non-adherent leukocytes from the blood vessels. Mice were then immediately perfused with fluorescein isothiocyanate (FITC)-conjugated Concanavalin A (20 μg/mL in 5 mL of PBS, pH 7.4; Vector Labs, Burlingame, CA, USA), as previously described [51 (link)], to label adherent leukocytes and vascular endothelial cells. After the eyes were removed and fixed in formalin, retinas were flat-mounted, prepared for quantification [52 (link)], and examined with the Axioskop microscope, and the images were digitized. The total numbers of leukocytes adhering to the retinal vessels were counted with the investigator being masked as to the nature of the specimen.

At 7 days after laser injury, the mice were perfused transcardially with phosphate-buffered saline (PBS) and then with FITC-conjugated concanavalin A (20 μg/mL in PBS; Vector Laboratories, Burlingame, CA) to label the CNVs. The eyes were removed and the posterior segment of the eyeball was prepared as a flatmount. RPE flatmounts were observed using a fluorescence microscope (Keyence Corporation, Tokyo, Japan). Measurements of CNV lesion area were performed using ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsbweb.nih.gov/ij/index.html). The outline of the CNV was drawn around the perimeter of the lesion and then total lesion area was measured46 (link). The average CNV area obtained from 4 lesions in each eye was used to give a single value for analysis.

Retinal leukostasis was performed as described previously [10 ]. Mice were anesthetized by isoflurane inhalation and perfused through the left heart ventricle with 10 ml phosphate-buffered saline (PBS) to remove erythrocytes and non-adherent leukocytes. The mice were perfused with fluorescein isothiocyanate (FITC)-conjugated concanavalin A (Vector Laboratories, Burlingame, CA, USA) to label adherent leukocytes, followed by 10 ml PBS perfusion to flush out unbound concanavalin A. The eyes were fixed in 4% paraformaldehyde for 1 h and the retinas were flat mounted. Luminal leukocytes were counted by an investigator blinded to different groups. Total number of adherent leukocytes in vessels per retina was statistically analyzed.

All experiments were approved by the Vanderbilt University Institutional Animal Care and Use Committee and were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Six-week old C57BL/6J mice (Charles Rivers; Wilmington, MA) were injected intravitreally with 2 μl of vehicle (0.1% DMSO and 0.3% EtOH), TNFα (50 ng/ml), TNFα with 11,12-EET (0.5 μM) plus AUDA (10 μM), or TNFα with 19,20-EDP (0.5 μM) plus AUDA (10 μM). As described previously11 (link), 6 hours after treatment mice were anesthetized with ketamine and xylazine and perfused with 0.9% saline for 1 minute, followed by FITC-conjugated concanavalin-A (40 μg/ml in 2.5 ml PBS; Vector Laboratories; Burlingame, CA). Mice were then perfused with saline for 5 minutes to remove any non-adherent leukocytes. Retinas were immediately dissected into 4% paraformaldehyde, flat-mounted, and images were captured with an AX70 upright microscope (Olympus; Tokyo, Japan) and DP71 digital camera (Olympus) at 4x magnification. Adherent leukocytes in the vasculature were counted and divided by the total retinal vascular area.