Gsl 1 isolectin b4

Retinal flat-mounts were prepared with modification to a previously published method [19 (link)]. In brief, the fixed eyes were washed in ice cold 1× PBS for 2 min and retinas were dissected in 1–2 ml of ice cold 1× PBS under a dissection microscope. The fully dissected retinas were washed twice in 1× PBS prior to staining. Fixed retinas were permeabilized for 2 h in PBS-BSA-Triton-X (1× PBS, 1% BSA, 1% Triton-X) at room temperature followed by washing in Wash Buffer (1× PBS, 0.5% Triton-X) for five times. Retinas were incubated overnight in the dark at 4°C with biotinylated GSL-1B4 (1:100 in Wash Buffer) with specificity for α-galactosylated glycoprotein residues on vascular endothelial cells and macrophages (Griffonia Simplicifolia Lectin I (GSL I) isolectin B4; Catalog#: B-1205-.5, Biotinylated; Vector Laboratories, Burlingame, CA, U.S.A.). Retinas were washed five times in 1× PBS and secondary antibody Cy3-Streptavidin (1:500 in 1× PBS; GE Healthcare, Amersham, U.K.) was added and incubated for 5 h in the dark at room temperature. Stained retinas were washed five times in 1× PBS, mounted and coverslipped with VECTASHIELD HardSet Anti-fade Mounting Medium (Vector Laboratories, Burlingame, CA, U.S.A.).