Csu x1 spinning disk unit

The aortic ring assay was performed according to a previously published protocol (44 ). Briefly, after euthanasia of WT or Del-1^−/− mice, the mouse aorta was dissected, flushed with serum-free Opti-MEM medium, cleared from surrounding adipose tissue, cut into small rings (each approximately 0.5 mm long) and incubated for 24 hours in serum-free Opti-MEM medium in a humidified incubator at 37°C and 5% CO₂. The rings were subsequently incubated in collagen-containing gels with Opti-MEM medium supplemented with 2.5 % FBS in the absence or presence of VEGF (15 ng/ml) at 37°C and 5% CO₂. After 5 days the plate was washed and fixed with 4% PFA. After permeabilization with 0.25% Triton X-100, BS1 lectin-FITC (0.1 mg/ml) was added to stain the endothelium for 24 hours at 4°C. After washing, Z-stack images were taken using a Leica LSI Macro confocal microscope and the individual images were analysed for the number of microvessels with the help of Image J software. Images of aortic rings displayed in the figure were acquired with an inverted Olympus IX 83 spinning disk microscope equipped with a Yokogawa CSU-X1 Spinning Disk Unit.

WT and Del-1–deficient (Del-1^−/−) mice were sacrificed either on postnatal day 6 (P6) in order to assess physiological retinal vascularization or were subjected to the ROP model and sacrificed on day P15. For retina whole mounts, the eyes were enucleated and fixed in 4% PFA at 4°C overnight. The next day, the retinas were carefully removed and permeabilized for 1h at RT in PBS with 1% BSA and 0.5% TritonX-100 and then incubated in FITC conjugated isolectin B4 (10 μg/ml, Bandeiraea simplicifolia; Sigma-Aldrich, Germany) at 4°C overnight. For the analysis of CD45⁺ positive cells in the retina, whole mounts were additionally stained with PE-conjugated anti-CD45 antibody or isotype control (1:50, BD Pharmingen). After flat mounting the retinas were imaged with an Axiovert 200 Inverted Fluorescence Microscope and Axiovision image processing software (Zeiss, Germany). The assessment of the vascular area was performed with Axiovision software (Zeiss Germany).
The expression of Del-1 in the retina was analysed by staining for β-Galactosidase (β-Gal) in cross-sections of Del-1–LacZ knock-in mice (or WT mice as control) together with a rat anti-CD31 antibody (PharmingenTM, Germany) to identify vessels. Staining was performed as described (13 (link)). Images were acquired with an inverted Olympus IX 83 spinning disk microscope equipped with a Yokogawa CSU-X1 Spinning Disk Unit.