Ab50009

The retinas were isolated and fixed in cold 4% PFA for 15 min, washed in PBS for 5-10 min, and blocked in 1% TritonX-100/5% BSA for 30 min at 37 °C. The whole-mount retinas were stained with NG2 antibody (1:100, ab50009, Abcam, USA) overnight at 4 °C. The primary antibody was removed and 1 × PBST (0.1% Tween-20 in 1×PBS, pH 7.4) was used to wash the retinas 4 × 10 min. The retinas were then stained with the Alexa Fluor 594 goat anti-mouse IgG (1:500, A11005, Invitrogen, USA) for 3 h at room temperature to label pericytes. Endothelial cells were labeled by staining Isolectin B4 (IB4, 1:100, L2895, Sigma, USA) for 2 h at room temperature. The staining signaling was captured using a fluorescent microscope (IX73P1F, Olympus, Japan).

Pericytes were labelled by expression of DsRed under control of the NG2 promoter (in mice), or with antibodies to NG2 (1:200; Abcam ab50009, Cambridge, United Kingdom), α-smooth muscle actin (α-SMA) (1:100; Abcam ab5694, Cambridge, United Kingdom), or myosin light chain (phospho S20, 1:100, Abcam ab2480, Cambridge, United Kingdom), and the capillary basement membrane and pericytes were labelledwith isolectin B₄-Alexa Fluor 647 (1:200, overnight; Molecular Probes, I32450, Thermo Fisher Scientific, Waltham, MA). Z-stacks of the cortex and outer medulla (frame size 640.17 × 640.17 µm) for cell counting were acquired confocally (Zeiss LSM 700, Oberkochen, Germany). Pericyte intersoma distance was calculated between pairs of pericytes on capillaries within the same imaging plane. Kidney damage was assessed using kidney injury molecule-1 (Kim-1) antibody (1:100, overnight; Novus Biologicals, NBP1-76701, Abingdon, United Kingdom). Red blood cells were labelled with antibody to glycophorin A (1:2000, AbCam ab9520, Cambridge, United Kingdom). Alexa Fluor conjugated secondary antibodies were added overnight (1:500; ThermoFisher, A31572, A31556, A31570, Waltham, MA).

Confocal microscopy was performed to confirm an increased BBB permeability to Evans Blue dye. Rats were decapitated and the brains were quickly removed, fixed with 4% neutral buffered formalin for 24 h, and cut into 50-µm thick slices on a vibratome (Leica VT 1000S Microsystem, Germany). The slices were incubated for one night with the goat anti-mouse NG2 antibody (1:500; ab 50009, Abcam, Cambridge, United Kingdom) and goat anti-rabbit GFAP antibody (1:500; ab 207165, Abcam, Cambridge, United Kingdom). In the next day, after several rinses in phosphate-buffered saline, the slices were incubated for 1h with 130 µl fluorescent-labeled secondary antibodies (goat anti-mouse IgG (H+L) Alexa Four 647; goat anti-rabbit IgG (H+L) Alexa Four 488; Invitrogen, Molecular Probes, Eugene, Oregon, USA). The slices were analyzed using a confocal microscope (Leica SP5, Germany). Approximately 8–12 slices per animal from cortical and subcortical (excepting hypothalamus and choroid plexus where the BBB is leaky) regions were imaged.

After the pups were humanly sacrificed, the eyeballs were harvested. Retinas were acquired according to previous study [19 ]. Fixation and dehydration were accomplished with 4% paraformaldehyde (PFA) and 2×phosphate-buffered saline (PBS), respectively. The retina was acquired by discarding the anterior and posterior segments. Before staining, the retina was incubated with methanol and stored at −20°C to increase permeabilization. Staining was conducted according to the manufacturer's protocol. Isolectin-B4 was purchased from Sigma-Aldrich LLC, and primary antibody of NG2 (ab50009) and secondary antibody against mouse (ab150115) were purchased from Abcam plc. Retinas were photographed using a confocal microscopy (Fluoview 1000, Olympus, Tokyo, Japan). In Isolectin-B4 staining, the superficial vascular plexus (SVP) was presented with green, and the inner and outer deep vascular plexus (iDVP, oDVP) were yellow and purple, respectively. A comprehensive illustration of the retinal vasculature was generated and assembled using Adobe Photoshop and Adobe Illustrator (Adobe Systems, San Jose, CA, USA). In Isolectin-B4 and NG2 double staining, vessels were shown as green and NG2 was red.

The mixtures of CECs and pericytes (at a ratio of 1:1) were suspended in the complete medium and seeded onto the Matrigel-precoated 24-well plate, then incubated at 37 °C in a humidified atmosphere of 5% CO₂ for up to 12 h. The harvested cells were fixed in 4% paraformaldehyde for 15 min, washed with PBS, and permeabilized with PBS containing 0.1% Triton X-100 for 30 min. These cells were stained with CD31 (1:200; 550300, BD Pharmingen) and NG2 (1:100; ab50009, Abcam) to identify ECs and pericytes, respectively. The images were observed under an IX73P1F fluorescent microscope (Olympus, Tokyo, Japan). Image J software was used to calculate pericyte coverage.

The sprouting choroidal tissue was used for detecting pericyte coverage by immunofluorescence staining. The choroidal explants were gently washed with ice-cold PBS, fixed with 4% paraformaldehyde for 15 min at room temperature, and blocked with 0.1% Triton X-100/3% BSA for 30 min at 37 °C. Pericytes were labeled with NG2 (1:100; ab50009, Abcam) overnight at 4 °C and Alexa Fluor 594 goat anti-mouse IgG (1:500; A11005, Invitrogen) for 3 h at room temperature. ECs were labeled with Isolectin GS-IB4 (1:100; L2895, Sigma, USA) for 2 h at room temperature. The images were captured under a fluorescence microscope (Olympus, Tokyo, Japan). NG2 positive area was measured using the Image J software.