Leukostasis

Retinal leukostasis assay, which allows labeling of leukocytes adherent to the retinal endothelium, was performed as described previously (Liu et al., 2019; Rojas et al., 2010). This assay has been widely used to study retinal vascular inflammation in mouse models of retinopathies (Al-Shabrawey et al., 2008; Chen et al., 2013; Gubitosi-Klug et al., 2008; Liu et al., 2019; Moore et al., 2003; Rojas et al., 2010). Briefly, 24 h after IR procedure, mice were deeply anesthetized by i.p. injection of a mixture of 100 mg/kg ketamine hydrochloride and 10 mg/kg xylazine hydrochloride. After the chest cavity was carefully opened, a 20-gauge perfusion cannula was introduced into the aorta, and mice were perfused with 10 ml PBS through the left ventricle of the heart to remove nonadherent blood cells. Next, 10 ml FITC-conjugated Con A lectin (40 µg/ml in PBS, pH 7.4; RL-1002, Vector Laboratories) was perfused to label the adherent leukocytes and vasculature, followed by 10 ml PBS perfusion to remove the residual unbound Con A. After eyeballs were collected and fixed with 4% paraformaldehyde (PFA) overnight, retinas were dissected and stained with anti-CD45 antibody (1:400, 550539, BD Biosciences). Leukocytes inside the blood vessels (leukostasis) are Con A⁺, CD45⁺ (green and red fluorescence), while leukocytes outside the blood vessels (leukocytes infiltrated into the retina) are Con A⁻, CD45⁺ (only red fluorescence). The total number of the adherent leukocytes per retina and leukocytes infiltrated into the retina per field were counted.