Multiplexed 3D virtual histology of intact murine meibomian glands in combination with immunofluorescence and HCR-FISH

3D virtual histology (3DVH) enables reconstruction and analysis of tissues in their intact three-dimensional state, which is unachievable by gold-standard 2D histology. Pairing 3DVH with immunofluorescence (IF) and fluorescence in situ hybridization (HCR-FISH) would enable combined visualization of tissue structure and molecular expression, but this approach is not well-established. Ocular pathologies like meibomian gland (MG) dysfunction affect complex 3D tissue morphology and molecular function concurrently. Here, we demonstrate 3DVH IF/HCR-FISH to visualize neural control of MGs in healthy and disease states, highlighting the need for better 3D structure-function visualization of ocular structures to understand pathology and guide treatment development.