An optimized click chemistry method allows visualization of proliferating neuronal progenitors in the mouse brain

We establish a click reaction-based workflow (tissue clearing coupled with click-chemistry in 3D, C⁴-3D) to visualize 5-ethynyl-2'-deoxyuridine (EdU) in whole mouse brain tissue cleared by CUBIC, iDisco+, and PACT. C⁴-3D was compatible with immunostaining, nuclear staining, and a fluorescent reporter mouse. Machine learning-based identification of EdU-positive nuclear coordinates followed by normalization for the Allen Brain Atlas revealed that proliferating neuronal progenitors were enriched in the subventricular zones (SVZs) and in their migration pathways to the olfactory bulbs and were decreased with aging. C⁴-3D for EdU was also applied to mouse models of cerebral infarction, glioblastoma multiforme, and metastatic brain tumor, as well as to kidney, liver, lung, and embryo in normal mouse. C⁴-3D will enable the exploration of cellular proliferation profiles in 3D. Especially, timed pulse-chase of EdU in normal development, disease progression, and tissue repair coupled with immunostaining will disclose the spatiotemporal generation, migration, and differentiation of newly synthesized cells.