Intracellular cholesterol visualization in brain tissue using D4H.

Studying cholesterol biology in the brain has been greatly hindered by the lack of adequate cholesterol visualization techniques. Here, we present a protocol for using a high-affinity cholesterol probe D4H-mCherry as a histology reagent in mouse or human brain tissue. We describe steps for D4H tissue treatment and crosslinking leading to stable labeling of intracellular membrane cholesterol. Furthermore, co-labeling with Rab5 endosomal marker and optimized buffers to reduce background enable punctate cholesterol visualization within the organelle membranes.