, Insets detail the signal taken at different extremities of the 3D volume, close (i or i') or away (iii or iii') from the imaging and illumination sides. (B) Alternative single labeling of the vessels using rat anti-Podocalyxin only. While this strategy offers the maximal flexibility for counter stains, the signal in the lateral thalamus is very low, but still segmentable (highlighted inset). (C) Alternative double labeling of the vessels against CD31 and Podocalyxin with goat-raised antibodies (D) Alternative triple immunolabeling for the vessels (CD31, Collagen IV and Podocalyxin) further improves the labeling gradient over the dual labeling (CD31, Podocalyxin) and has the best signal to noise ratio, while being less flexible for counter stainings. (E) Validation of the whole-mount passive immunolabeling. Brains whole-mount immunolabeled with the iDISCO+ protocol with primary (Podocalyxin) and secondary rounds of staining, then vibratome sliced and re-stained for CD31 on sections (n = 3). A thalamic section (the most challenging region of the brain in whole-mount stainings), is shown with several insets of different representative regions. Whole-mount and on-section immunolabels overlap perfectly in all regions inspected, surface, deep, as well as in gray and white matter, Comparison of the tissue and vascular integrities after iDISCO+ processing: coronal 100mm sections of perfused brains (left) or iDISCO+ processed brains (right), immunostained for Neurofilment M and CD31