Sample staining and iDISCO+ clearing

Whole-brain vasculature staining was performed following the iDISCO+ protocol previously described (Renier et al., 2016), with minimal modifications. All steps of the protocol were done at room temperature with gentle shaking unless otherwise specified. All buffers were supplemented with 0.01% sodium azide (Sigma-Aldrich) to prevent bacterial and fungal growth. Brains were dehydrated in an increasing series of methanol (Sigma-Aldrich) dilutions in water (washes of 1 h in methanol 20, 40, 60, 80, and 100%). An additional wash of 2 h in methanol 100% was done to remove residual water. Once dehydrated, samples were incubated overnight in a solution containing a 66% dichloromethane (Sigma-Aldrich) in methanol and washed twice in methanol 100% (4 h each wash). Samples were then bleached overnight at 4°C in methanol containing 5% hydrogen peroxide (Sigma-Aldrich). Rehydration was done by incubating the samples in methanol 60, 40, and 20% (1 h each wash). After methanol pretreatment, samples were washed in PBS twice, for 15 min and 1 h, in PBS containing 0.2% Triton X-100 (Sigma-Aldrich) and further permeabilized by 24-h incubation at 37°C in Permeabilization Solution (20% DMSO [Sigma-Aldrich] and 2.3% glycine [Sigma-Aldrich] in PBS-T).

To start the immunostaining, samples were first blocked with 0.2% gelatin (Sigma-Aldrich) in PBS-T for 24 h at 37°C, and the same blocking buffer was used to prepare antibody solutions. A combination of primary antibodies targeting different components of the vessel’s walls were used to achieve continuous immunostaining. Antibodies to podocalyxin and CD31 were combined with antibodies against the nucleocapsid (N) from GeneTex. Primary antibodies were incubated for 10 d at 37°C with gentle shaking, then washed in PBS-T (twice for 1 h and then overnight) and finally newly incubated for 10 d with secondary antibodies. Secondary antibodies conjugated to Alexa Fluor 647 were used to detect podocalyxin and CD31, while the NUCLEOCAPSID protein was stained with a secondary antibody conjugated to Alexa Fluor 555. After immunostaining, the samples were washed in PBS-T (twice for 1 h and then overnight), dehydrated in a methanol/water increasing concentration series (20, 40, 60, 80, and 100%, 1 h each, and then methanol 100% overnight), followed by a wash in 66% dichloromethane and 33% methanol for 3 h. Methanol was washed out with two final washes in dichloromethane 100% (15 min each), and finally the samples were cleared and stored in dibenzyl ether (Sigma-Aldrich) until light sheet imaging.