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Lookup NU author(s): Jonathan Phillips, Dr Alex LaudeORCiD, Professor Robert Lightowlers, Dr Christopher Morris, Emeritus Professor Doug Turnbull, Dr Nichola Lax
This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).
CLARITY enables immunofluorescent labelling and imaging of large volumes of tissue to provide a better insight into the three dimensional relationship between cellular morphology and spatial interactions between different cell types. In the current study, we optimise passive CLARITY and immunofluorescent labelling of neurons and mitochondrial proteins in mouse and human brain tissues to gain further insights into mechanisms of neurodegeneration occurring in mitochondrial disease. This is the first study to utilise human cerebellum fixed in paraformaldehyde and cryoprotected in conjunction with formalin-fixed tissues opening up further avenues for use of archived tissue. We optimised hydrogel-embedding and passive clearance of lipids from both mouse (n = 5) and human (n = 9) cerebellum as well as developing an immunofluorescent protocol that consistently labels different neuronal domains as well as blood vessels. In addition to visualising large structures, we were able to visualise mitochondrial proteins in passively cleared tissues to reveal respiratory chain deficiency associated with mitochondrial disease. We also demonstrate multiple use of tissues by stripping antibodies and re-probing the cerebellum. This technique allows interrogation of large volumes intact brain samples for better understanding of the complex pathological changes taking place in mitochondrial disease.
Author(s): Phillips J, Laude A, Lightowlers R, Morris CM, Turnbull DM, Lax NZ
Publication type: Article
Publication status: Published
Journal: Scientific Reports
Year: 2016
Volume: 6
Online publication date: 16/05/2016
Acceptance date: 20/04/2016
Date deposited: 05/07/2016
ISSN (electronic): 2045-2322
Publisher: Nature Publishing Group
URL: http://dx.doi.org/10.1038/srep26013
DOI: 10.1038/srep26013
PubMed id: 27181107
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