Eyes were fixed in 4% PFA in PBS at 4°C overnight and washed in PBS. Retinas were dissected, permeabilized in PBS, 1% BSA, and 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 (Bandeiraea simplicifolia; L-2140; Sigma-Aldrich) 20 μg/ml in PBlec at 4°C overnight. After five washes in PBS, samples were incubated with streptavidin conjugates (Alexa 488, 568, or 633; Molecular Probes) diluted 1:100 in PBS, 0.5% BSA, and 0.25% Triton X-100 at 4°C for 6 h. TO-PRO 3 (1:1,000; Molecular Probes) served for nuclear staining. After washing and a brief postfixation in PFA, the retinas were either flat mounted using Mowiol/DABCO (Sigma-Aldrich) or processed for multiple labeling. The following antibodies were used: GFAP (1:75; Z 0334; Dako), VEGFR2 (1:50; 555307; BD PharMingen), VEGFR2 (1:50; AF644; R&D Systems), F4/80 (1:100; MCAP497; Serotec), fibronectin (1:200; A 0245; Dako), VE-cadherin (1:1, culture supernatant provided by Dietmar Vestweber, University of Muenster, Muenster, Germany), and Ki67 (1:200; NCL-Ki67p; Novo Castra). Alexa-488, 568, or 633 conjugated secondary antibodies (Molecular Probes). Rhodamine-phalloidin served for actin staining (1:40; Molecular Probes). VEGFR2 signal was amplified using the TSATM Fluorescein System (NEL701; NEN) according to instructions. Flat mounted retinas were analyzed by fluorescence microscopy using a Nikon E1000 microscope equipped with a digital camera (Nikon Coolpix 990) and by confocal laser scanning microscopy using a Leica LCS NT. Images were processed using Adobe Photoshop®.
For visualization of vascular lumina, FITC-conjugated Dextran (FD-2000S; Sigma-Aldrich) was warmed to 37°C and perfused through the heart of deeply anaesthetized mice (Avertin, intraperitoneally 10 μl/g body weight).
Mouse VEGF-A, PDGF-B, VEGFR1, and VEGFR2 cDNA fragments were used for whole mount in situ hybridization as described (Fruttiger, 2002 (link)). For double labeling, immunolabeling was performed after a 10-min postfixation in 4% PFA.
BrdU labeling was achieved by a 2-h BrdU pulse before fixation (100 μg Brd U/g body weight, intraperitoneally). For double labeling, isolectin labeling was followed by a 30 min 4% PFA fixation, three washes in PBS, a 1-h incubation in 6 M HCl and 0.1% Triton X-100, six washes in PBT, blocking, and anti-BrdU antibody (1:50, 347580; BD PharMingen) incubation.
For visualization of vascular lumina, FITC-conjugated Dextran (FD-2000S; Sigma-Aldrich) was warmed to 37°C and perfused through the heart of deeply anaesthetized mice (Avertin, intraperitoneally 10 μl/g body weight).
Mouse VEGF-A, PDGF-B, VEGFR1, and VEGFR2 cDNA fragments were used for whole mount in situ hybridization as described (Fruttiger, 2002 (link)). For double labeling, immunolabeling was performed after a 10-min postfixation in 4% PFA.
BrdU labeling was achieved by a 2-h BrdU pulse before fixation (100 μg Brd U/g body weight, intraperitoneally). For double labeling, isolectin labeling was followed by a 30 min 4% PFA fixation, three washes in PBS, a 1-h incubation in 6 M HCl and 0.1% Triton X-100, six washes in PBT, blocking, and anti-BrdU antibody (1:50, 347580; BD PharMingen) incubation.
