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.
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.
