Te ez c1 confocal system

Assessment of the BRB was conducted in cryosections, by assessing albumin leakage from the retinal vasculature. The vasculature was visualised using biotinylated isolectin GS-IB4 (Sigma) followed by binding to streptavidin–Alexa Fluor 488 (Life Technologies, Paisley, UK). Albumin was localised using a goat anti-mouse albumin antibody (Bethyl Laboratories, Montgomery, TX, USA) followed with anti-goat Alexa Fluor 568 (1:500; Life Technologies). Vessel or neuropile-localised fluorescence was visualised by using a Nikon TE EZ-C1 confocal system (Nikon, Kingston Upon Thames, UK). Using NIS Elements software (Nikon) a threshold value of 12,000 was set for measuring image intensity/brightness. The area (μm²) of the albumin staining was measured with the 12,000 brightness intensity threshold in the retinal sections. Images were taken at three separate points on the central retina at magnification ×40 and n = 5 per group of mice.

Leucostasis in diabetic mice was assessed after 4 weeks of diabetes using fluorophore-conjugated concanavalin-A, as described previously [16 (link)]. The retinas were mounted on slides and visualised using a Nikon TE EZ-C1 confocal system (Nikon). Two z stacks of the retina for each artery/vein with the capillaries (central and peripheral) were taken and the total number of leucocytes in the arteries, veins and capillaries were counted.

Following treatments, living cells were incubated for 1 h with caspase 1 probe, FAM-YVAD-FMK, according to manufacturer’s instructions (Immunochemistry Technologies). Alternatively, cells were incubated for 0.5 h with 2.5 μM acridine orange (Thermofischer Scientific) to label acidic lysosomal compartments. After being washed in PBS twice, cells were fixed in 4 % Paraformaldehyde in PBS for 20 mins, mounted with ProLong Gold Antifade Mountant containing DAPI (Invitrogen) and visualised using a Nikon TE EZ-C1 confocal system (Nikon, Kingston Upon Thames). Images were captured with a 60 × objective lens and a 1024 × 1024 for the caspase 1 probe and a 512 × 512 frame for the lysosomal staining.