Rabbit anti collagen 4

Retinas were dissected and fixed as described previously (46 (link)) and then whole-mount stained with rabbit anti–collagen IV (Bio-Rad, 2150-1470), Alexa Fluor 488–conjugated anti-rabbit IgG (Thermo Fisher Scientific, A11008) or biotin-conjugated isolectin B4 (IB4) (Sigma-Aldrich, L2140), and Alexa Fluor 647–conjugated streptavidin (Thermo Fisher Scientific, S21374), which were all diluted in PBS containing 10% serum-free protein block (Dako) and 0.1% Tween-20. Dissected corneas were fixed in 2% formaldehyde for 1 hour and then whole-mount stained with Cy3-conjugated neuron-specific beta-III tubulin (R&D Systems, NL1195R) or FITC-conjugated rat anti–mouse PECAM-1 (CD31) (BD Biosciences, 558738). Antibodies were diluted in PBS containing 3% BSA and 0.1% Triton X-100. Staining was visualized with an Eclipse Ti inverted epifluorescence microscope (Nikon).

Mesenteries were fixed in 4% paraformaldehyde at room temperature for 2 h and stained as previously described (Stanczuk et al., 2015 (link)). The following primary antibodies were used: mouse anti-α-smooth muscle actin-Cy3 (Sigma-Aldrich, C6198, 1:250), rat anti-endomucin (V.7C7) (Santa Cruz, SC-65495, 1:200), rat anti-CD41-FITC (eBioscience, 11-0411-81, 1:50), rabbit anti-collagen IV (Bio-Rad, 2150-1470, 1:500), chicken anti-GFP (Abcam, ab13970, 1:200), goat anti-neuropilin 2 (R&D Systems, AF567, 1:200), hamster anti-PECAM1 (Millipore, MAB1398Z, 1:1000), rat anti-PECAM1 (BD Pharmingen, 553370, 1:1000), rat anti-PECAM1-AF594 (BioLegend, 102520, 1:100), rabbit anti-human PROX1 (Stanczuk et al., 2015 (link); 1:200), rat anti-TER-119 (eBioscience, 145921, 1:200), rat anti-TER-119-AF647 (BioLegend, 116218, 1:50), goat anti-VE-cadherin (Santa Cruz, SC-6458, 1:200) and anti-podoplanin (Developmental Studies Hybridoma Bank at the University of Iowa, clone 8.1.1, 1:800). Autofluorescence signal at 550-600 nm wavelength was used for visualization of RBCs. Secondary antibodies conjugated to AF488, AF594, AF647 or Cy3 were from Jackson ImmunoResearch, and all were used at 1:300.

Brain sections were blocked with 0.5% TritonX-100 and 10% donkey serum in PBS for 1 h at room temperature, after which the sections were incubated with primary antibodies overnight at 4°C. Primary antibodies included rat anti-CD31 (antiplatelet endothelial cell adhesion molecule-1, labels blood vessels) (Xu et al., 2017 (link)) (BD Pharmingen, 553369), rabbit anti-desmin (Cell Signaling, #5332), and rabbit anti-collagen IV (Bio-Rad, 2150-1470). The slides were washed and then incubated with Alexa Fluor 488-conjugated species-appropriate secondary antibody (Vector Labs, DI-1488) and Cy3-conjugated streptavidin (Invitrogen, A10522) for 1 h at room temperature in the dark. Then, the slides were stained with 4′,6-diamidino-2-phenylindole for 5 min in the dark, and the brain slices were washed with PBS, then patched and mounted.