Next generation histology methods for three-
dimensional imaging of fresh and archival human
Hei Ming Lai
, Alan King Lun Liu
, Harry Ho Man Ng
, Marc H. Goldﬁnger
, Tsz Wing Chau
, Bension S. Tilley
, Wai Man Wong
, Wutian Wu
& Steve M. Gentleman
Modern clearing techniques for the three-dimensional (3D) visualisation of neural tissue
microstructure have been very effective when used on rodent brain but very few studies have
utilised them on human brain material, mainly due to the inherent difﬁculties in processing
post-mortem tissue. Here we develop a tissue clearing solution, OPTIClear, optimised for
fresh and archival human brain tissue, including formalin-ﬁxed parafﬁn-embedded material. In
light of practical challenges with immunostaining in tissue clearing, we adapt the use of cresyl
violet for visualisation of neurons in cleared tissue, with the potential for 3D quantiﬁcation in
regions of interest. Furthermore, we use lipophilic tracers for tracing of neuronal processes in
post-mortem tissue, enabling the study of the morphology of human dendritic spines in 3D.
The development of these different strategies for human tissue clearing has wide applicability
and, we hope, will provide a baseline for further technique development.
School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong 852, Hong Kong.
Neuropathology Unit, Division
of Brain Sciences, Imperial College London, Burlington Danes Building, Hammersmith Campus, Du Cane Road, London W12 0NN, UK.
GMH Institute of
CNS Regeneration, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China.
Re-Stem Biotechnology Co., Ltd, 2588 Wuzhong Road, Suzhou
215310, China. These authors contributed equally: Hei Ming Lai, Alan King Lun Liu and Harry Ho Man Ng. Correspondence and requests for materials should
be addressed to W.W. (email: email@example.com) or to S.M.G. (email: firstname.lastname@example.org)