Sodium Cholate-Based Active Delipidation for Rapid and Efficient Clearing and Immunostaining of Deep Biological Samples.

Recent surges of optical clearing provided anatomical maps to understand structure-function relationships at organ scale. Detergent-mediated lipid removal enhances optical clearing and allows efficient penetration of antibodies inside tissues, and sodium dodecyl sulfate (SDS) is the most common choice for this purpose. SDS, however, forms large micelles and has a low critical micelle concentration (CMC). Theoretically, detergents that form smaller micelles and higher CMC should perform better but these have remained mostly unexplored. Here, SCARF, a sodium cholate (SC)-based active delipidation method, is developed for better clearing and immunolabeling of thick tissues or whole organs. It is found that SC has superior properties to SDS as a detergent but has serious problems; precipitation and browning. These limitations are overcome by using the ion-conductive film to confine SC while enabling high conductivity. SCARF renders orders of magnitude faster tissue transparency than the SDS-based method, while excellently preserving the endogenous fluorescence, and enables much efficient penetration of a range of antibodies, thus revealing structural details of various organs including sturdy post-mortem human brain tissues at the cellular resolution. Thus, SCARF represents a robust and superior alternative to the SDS-based clearing methods and is expected to facilitate the 3D morphological mapping of various organs.

Vaccinia-related kinase 2 plays a critical role in microglia-mediated synapse elimination during neurodevelopment.

During postnatal neurodevelopment, excessive synapses must be eliminated by microglia to complete the establishment of neural circuits in the brain. The lack of synaptic regulation by microglia has been implicated in neurodevelopmental disorders such as autism, schizophrenia, and intellectual disability. Here we suggest that vaccinia-related kinase 2 (VRK2), which is expressed in microglia, may stimulate synaptic elimination by microglia. In VRK2-deficient mice (VRK2KO ), reduced numbers of presynaptic puncta within microglia were observed. Moreover, the numbers of presynaptic puncta and synapses were abnormally increased in VRK2KO mice by the second postnatal week. These differences did not persist into adulthood. Even though an increase in the number of synapses was normalized, adult VRK2KO mice showed behavioral defects in social behaviors, contextual fear memory, and spatial memory.