Mounting medium

ARPE19 cells were cultured on cover slips under different treatment conditions, including oxPOS and zinc depleted and zinc supplemented for 48 h. Cells were washed with cold PBS and fixed with 1% paraformaldehyde (PFA) for 10 min, followed by permeabilization with 0.1% Triton-X for 5 min. The samples were then blocked with 5% BSA for 30 min at room temperature and incubated with anti-human cathepsin B (1 : 100, Life Technologies, UK) antibody for 2 h. After washing with PBS, samples were incubated with anti-mouse IgG 488 secondary antibody at a dilution of 1 : 100 with PBS and nuclei stained with DAPI at a dilution of 1 : 500 (Life Technologies, UK) in the dark for 1 h. Following thorough washing, coverslips were mounted with mounting medium (Vector Laboratories, Peterborough, UK) and examined by confocal microscopy (Eclipse TE2000-U, Nikon, Surrey, UK) at 40x magnification and numerical aperture at 0.65.
Dye-conjugated molecular probes used for staining intracellular organelles or other molecules used in this study are listed in Table 1. The dyes were added to live cells overnight in the dark at 37°C according to the manufacturers' instructions. After washing, fresh media was added and the samples were imaged by confocal microscopy.

2 ml of PBS containing 500 units of heparin were perfused through the heart of a deeply anesthetized mouse, followed by 1 ml PBS containing 15mg/ml FITC conjugated Dextran (Sigma) and 4% paraformaldehyde (PFA), prepared immediately prior to use. Eyes were post-fixed in 4% PFA in PBS overnight at 4°C and retinas were dissected, post-fixed in 4% PFA in PBS for 30min at RT and flat-mounted in Mounting medium (Vector Lab).

Unimmunized and IRBP-immunized mice (day 9 post-immunization) treated with either control or 4N1K peptide were anesthetized and perfused with 10 mL of PBS to remove intravascular content, including non-adherent leukocytes. Perfusion with 10 mL of Rhodamine-conjugated Concanavalin A (ConA, 5 µg/mL in PBS pH 7.4) (Vector labs, Burlingame, CA, USA) was performed to label adherent leukocytes and vascular endothelial cells. Residual unbound lectin was removed by perfusing an additional 10 mL of PBS. The carefully harvested retinas were flat mounted in a mounting medium (Vector labs). Each retina was imaged with fluorescence microscope (Olympus FSX100, Tokyo, Japan), and the adherent leukocytes per retina were counted. Similar assessments were performed on wild-type and TSP-1-deficient mice 24 h after injecting 100 µg of LPS from Salmonella typhimurium (Sigma Chemical, St. Louis, MO, USA) in one hind footpad.