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Development of passive CLARITY and immunofluorescent labelling of multiple proteins in human cerebellum: understanding mechanisms of neurodegeneration in mitochondrial disease

Lookup NU author(s): Jonathan Phillips, Dr Alex LaudeORCiD, Professor Robert Lightowlers, Dr Christopher Morris, Emeritus Professor Doug Turnbull, Dr Nichola Lax

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Abstract

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.

Publication metadata

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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