Ab769

PDGF-BB (220-BB, R&D Systems). Antibodies used were as follows: anti-NCK1 (1/200, ab168940, Abcam), anti-NCK2 (1/200, ab109239, Abcam), anti-PDGF-B (1/50, AB23914, Abcam), anti-PDGFRβ (1/100, AF1042, R&D Systems), anti-NG2 (1/200, AB5320, Millipore), anti-MYH11 (1/200, AB53219, Abcam), anti-DESMIN (1/200, AF3844, R&D Systems), anti-TER119 eFluor 450 (1/50, 48-5921-82, eBioscience-ThermoFisher Scientific), anti-fibronectin (1/300, F3648, Sigma-Aldrich), anti-laminin (1/300, AB11575, Abcam), anti-collagen IV (1/300, AB769, Millipore), anti-GFP (1/300, G10362, Life Technologies), anti-ERG1/2/3 (1/100, SC353, Santa Cruz), anti-phospho-histone 3 (1/100, 06-570, Millipore), anti-α-smooth muscle actin CY3 (1/200, α-SMA, C6198, Sigma), anti-PDGFRβ 751 (1/1000, #4549, Cell Signaling), anti-PDGFRβ 1009 (1/1000, #3124, Cell Signaling), anti-PDGFRβ 1021 (1/1000, #2227, Cell Signaling), anti-PDGFRβ (1/1000, #3169, Cell Signaling), anti-p44/42 MAP kinase (1/1000, phospho-ERK, #9106, Cell Signaling), anti-44/42 MAP kinase (1/1000, total ERK, #9102, Cell Signaling), anti-pAKT (1/1000, #4060, Cell Signaling), and anti-panAKT (1/1000, total AKT, #4685, Cell Signaling). Appropriate secondary antibodies were conjugated to horseradish peroxidase (Vector Laboratories) or fluorescently labeled (Life Technologies). IB4 was purchased from Life Technologies.

For fluorescence microscopy, the mice were perfused with PBS followed by 4% paraformaldehyde (PFA). After enucleation, eyes were fixed in 4% PFA for 1 h at 4 °C in the dark and retinal flatmounts were dissected prior to staining as previously described [34 (link),35 (link)]. Primary antibodies against IBA1 (AB178846, Abcam, Cambridge, United Kingdom) and Collagen IV (COL4, AB769, Merck Millipore, Billerica, MA, USA) were added over two nights at a dilution of 1:500 at 4 °C in the dark. Secondary antibodies were added at a dilution of 1:500 (Alexa Fluor^® 647 and Alexa Flour^® 488, Life Technologies, Carlsbad, CA, USA) overnight at 4 °C in the dark. Retinal flatmounts were imaged using a Hamamatsu NanoZoomer S60 (Hamamatsu Photonics, Herrsching, Germany). To quantify microglia cell numbers, confocal images of RAP lesions were taken using a Leica SP8 confocal microscope with a 20× objective lens (Leica, Wetzlar, Germany). RAP lesions were detected when focusing below the outer plexiforme layer and were defined as neovascularizations originating from retinal vessels and extending into the normal avascular outer nuclear layer and reaching the retinal pigmented epithelium.

Endothelial and pericyte-like cells (HUVECs/PVCs) were co-cultured in 2% collagen I gel. Cultures were incubated in ECGM2 medium supplemented with PDGF-BB and VEGF (10 ng ml⁻¹ each) for 6 days. Recombinant Net4-FL and Net4-FL^E195A,R199A were added to the three-dimensional gel solution at indicated concentration. Medium (supplemented with PDGF-BB, VEGF, and Net4-FL or Net4-FL^E195A,R199A) was replaced every 48 h. Samples were fixed with Dent's fixative and immunostained for collagen IV (1:500; AB769, Chemicon), laminin γ1 (1:500; kind gift from U. Mayer, University of East Anglia), and human CD31 (1:500; 555444, BD Biosciences). DNA was stained by Hoechst 33258. Overview pictures were taken from each sample by using a Zeiss Axioplan microscope and average tube length was calculated by the Volocity software. Optical section images were taken by a Zeiss Apotome microscope and processed by Axiovision program. All experiments were performed in groups of five independent cultures and repeated at least three times. Validation information for the laminin γ1 antibody is presented in the work of Mayer et al.64 (link)

Mouse hind paws were isolated after neurobehavioral tests. Dissected tissues were fixed with 10% paraformaldehyde overnight and cryoprotected for 72 h with 30% sucrose in PBS at 4 °C. The samples were embedded in OCT (#CM 3050S; Leica) and sliced into 30 μm thick sections. The slides were washed with 0.05% Triton X-100 in PBS for 4 h and incubated with CAS-Block^TM solution (#008120; Life Technologies) at room temperature for 1 h. The primary antibodies used were rabbit anti-PGP9.5 (1:100; #ab108986, Abcam) and goat anti-Collagens VI (1:100, #AB769; Chemicon). For double staining, two antibodies from different species were mixed and incubated overnight with the sample sections at 4 °C. After washing away the primary antibodies with PBS, the tissues were incubated with secondary antibodies (Alexa-488, 594, or 647, 1:200; Invitrogen) and Hoechst 33342 (10 mg/mL, #H1399; Invitrogen) for 2 h at room temperature. Immunofluorescence images were acquired using a multiphoton laser scanning microscope with a 40× objective (FV1000MPE, Olympus).