Biotinylated isolectin b4

Eyes were enucleated and fixed by immersion in 4% paraformaldehyde for 1 hour. Retinas were isolated by dissection, and flat mounted following incubation with biotinylated isolectin B4 (Sigma-Aldrich, 1/200 from 1mg/ml stock) and Alexa Fluor 546 conjugated streptavidin (Life Technologies). Images were taken by using a 10x objective and tilescan options. The extent of retinal vascular regression (central avascular area) and pre-retinal vascularization (NV) were measured using Image J [5 (link)], by quantification of manually selected area and intensity threshold- selected particles, respectively. For GFAP staining, eyes were fixed in 1% paraformaldehyde for 1 hour, embedded in OCT (RA Lamb) and frozen in isopentane precooled in liquid nitrogen. Sections of 18 μm of thickness were incubated with rabbit anti-GFAP antibody (Dako) and anti-rabbit-488 secondary antibody (Life Technologies). Lungs of the nursing mothers were dissected and fixed by immersion in 4% paraformaldehyde for 12 hours. DeadEnd^™ Fluorometric TUNEL System kit (Promega) was used to determine apoptosis in 10, non-consecutive lung cryosections. Alternatively, cryosections were incubated with anti-rat CD45 (Millipore) or Van Gieson stain.

For whole mount retina immunofluorescence, we proceeded as Pitulescu et al [42 (link)]. Eyes were fixed in 4% paraformaldehyde in PBS at 4°C overnight and washed in PBS. Retinas were dissected, permeabilized in PBS, 1% BSA, 0.5% Triton X-100 at 4°C overnight, rinsed in PBS, washed twice in PBlec (PBS, pH 6.8, 1% Triton-X100, 0.1 mM CaCl2, 0.1 mM MgCl2, 0.1 mM MnCl2), and incubated in biotinylated isolectin B4 (Sigma- Aldrich), 20 μg/ml in PBlec at 4°C overnight. After five washes in PBS, samples were incubated with streptavidin conjugates (Alexa-488, -594, or -647; Molecular Probes) diluted 1:400 in PBS, 0.5% BSA, 0.25% Triton X-100 at room temperature for 3 h. After washing and a brief postfixation in paraformaldehyde, the retinas were either flat-mounted using Prolong (Molecular 2 probes) or processed for multiple labeling. DAPI (1:5000, 20 mg/ml, Sigma) was used for nuclear staining. Flat-mounted retinas were analyzed by confocal laser scanning microscopy using a Nikon A1 microscope. Images were processed using ImageJ. Fields of views at the sprouting vascular front of the retinal vascular network, including regions of capillary-sized vessels directly adjacent to radial arterioles, were captured using a 40x objective lens. For quantification, at least four fluorescent images/retinas were taken from 8 mice per group.

Isolectin B4 staining was used to assess the purity and density of microglial cultures and the morphology of microglial cells. For this purpose, microglial cells were plated on poly-l-lysine-coated cover slips. After stimulation, cells were fixated in 4% formalin. Fixated cells were permeabilized with Triton X (0.1% in PBS) for 30 min and then incubated with biotinylated isolectin B4 (5 μg/ml, diluted in PBS + 1% BSA; Sigma-Aldrich, Taufkirchen, Germany) for 90 min. Thereafter, cells were treated with avidin-biotin complex (ABC, Vector, Burlingame, CA) for 30 min, and diaminobenzidine was used for visualization (5 min) resulting in a brown staining of the somata of microglial cells. The purity of microglia in the cultures was greater than 98%.