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.
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.
