I32450

Paraffin sections (10 μm) were incubated overnight with primary antibodies, followed by 1h incubation with a fluorescent-dye-conjugated secondary antibody. RBPJ and N1ICD staining was performed using tyramide signal amplification (PerkinElmer NEL744B001KT). CBF:H2B-Venus expression was detected using anti-GFP antibody. Antibodies used in this study are: anti-RBPJ (CosmoBio 2ZRBP2, 1:50), anti-Troponin T (DSHB CT3, 1:20) anti-Cleaved Notch1 ICD (Cell Signaling Technology 2421S, 1:100), anti-GFP (Aves Labs GFP-1010, 1:400), and anti-Myosin Heavy Chain MF-20 (DHSB, 1:20). DAPI (Sigma-Aldrich D9542, 1:1000) and Isolectin B4 glycoprotein (ThermoFisher I32450, 1:100). Confocal images were obtained using Leica SP5 confocal fluorescence microscope.

Eyeballs of P7 or P8 mice were fixed in 4% formaldehyde at 4 °C during 2 h and then washed in PBS and dissected. Retinas were permeabilized and blocked at 4 °C overnight in PBS, 1% BSA, 0.5% Triton X-100, 5% Normal donkey serum (Jackson ImmunoResearch). Ai3 and Ai14 retinas were incubated with isolectin B4 conjugated with Alexa Fluor-647 (IB4, ThermoFisher Scientific I32450, 1:200) in PBlec: PBS, 0,1 mM CaCl₂, 0,1 mM MgCl₂, 0,1 mM MnCl₂, 1% Triton X-100 pH 6.8 for 4 h at room temperature. R26R-EYFP and mTmG retinas were also incubated with chicken anti-GFP (Abcam ab13970, 1:500) during that time. This was followed by three 30′ washes in PBS, 0.5% BSA, 0.25% Triton X-100. R26R-EYFP and mTmG retinas were then incubated overnight at 4 °C with the Donkey anti-chicken 488 (Jackson ImmunoResearch, 703-545-155) in PBS, 0.5% BSA, 0,25% Triton X-100 and washed again three times 30′ in PBS. Retinas were mounted with ProLong Gold Antifade Mountant (Life Technologies, P36930) and imaged on a confocal laser-scanning microscope (Leica TCS SP8) with a 10X magnification objective.

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).