3D Imaging and Quantification of Lung Vasculature

The iDISCO (ace) procedure was performed according to the previously described protocol (Liu et al., 2020 (link)). The VE-cadherin antibody conjugated with Alexa Fluor™ 647 (VE-cadherin antibody, Biolegend, #138002. Alexa Fluor™ 647 dye, Thermo Fisher, #A20006) was diluted 1:1000 for use. After immunolabeling with Alexa Fluor dye-conjugated VE-Cadherin antibody, the tissues were washed directly with PBS/0.1% Tween 20/heparin (10 μg/ml) at room temperature for 24 h following the protocol described. The lung tissues processed by the iDISCO (ace) procedure were imaged on an Andor Dragonfly 200 Confocal Imaging System, with a ×10 objective, a step size of 8μm, 250 ms exposure time. About 1200-μm-thick optical stack of signal were acquired for each lung tissue. Imaris (version 9.9) was used to reconstruct the image stacks obtained from the confocal imaging to perform whole-tissue 3D assessment of the vasculature. AngioTool (version 0.6a) was used for vessels percentage area and total number of junctions analysis (Zudaire et al., 2011 (link)).