Dehydration and clearing of whole mouse brains and dissected hippocampi for ultramicroscopy

This protocol describes the preparation of whole mouse brains and dissected hippocampi for ultramicroscopy (UM), a powerful imaging technique that achieves precise and accurate three-dimensional (3D) reconstructions of intact macroscopic specimens with micrometer resolution. In UM, a specimen in the size range of ∼1-15 mm is illuminated perpendicular to the observation pathway by two thin counterpropagating sheets of laser light. Thus, specimens for UM need to be sufficiently transparent, which requires chemical clearing in most cases. In this protocol, mouse brains and hippocampi are carefully dissected and dehydrated, and then cleared in a solution of benzyl benzoate and benzyl alcohol.