Fig. 6.

Flowchart summarising the next-generation histology protocol developed in this paper. The protocol outlined here enables both fresh and archival formalin-fixed paraffin-embedded (FFPE) post-mortem tissues to be processed for tissue clearing. Formalin-fixed tissues can then be stained using immunohistochemical or non-immunohistochemical techniques such as lipophilic dye tracing with DiI or 3D chemical staining with cresyl violet. An optional SDS delipidation step is recommended before 3D immunostaining or chemical staining. Subsequently, tissues are rendered optically transparent with refractive index homogenization using OPTIClear before imaging or proceeding to other studies such as electron microscopy. H&E, haematoxylin & eosin; RI, refractive index; SDS, sodium dodecyl sulphate; TEM, transmission electron microscopy