title: Development of passive CLARITY and immunofluorescent labelling of multiple proteins in human cerebellum: understanding mechanisms of neurodegeneration in mitochondrial disease.
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.
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, NE2 4HH.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Phillips
authors.affiliation: Bioimaging Facility, Newcastle University, Newcastle upon Tyne, NE2 4HH, United Kingdom.
authors.forename: Alex
authors.initials: A
authors.lastname: Laude
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, NE2 4HH.|Institute for Cell and Molecular Biosciences Newcastle University, Newcastle upon Tyne, NE2 4HH, United Kingdom.
authors.forename: Robert
authors.initials: R
authors.lastname: Lightowlers
authors.affiliation: Medical Toxicology Centre, Newcastle University, Wolfson Building, Claremont Place, Newcastle NE2 4AA.
authors.forename: Chris M
authors.initials: CM
authors.lastname: Morris
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, NE2 4HH.
authors.forename: Doug M
authors.initials: DM
authors.lastname: Turnbull
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, NE2 4HH.
authors.forename: Nichola Z
authors.initials: NZ
authors.lastname: Lax
mesh_terms: D000368:Aged; D000818:Animals; D001808:Blood Vessels / Q000378:metabolism* / Q000473:pathology; D002531:Cerebellum / Q000378:metabolism* / Q000473:pathology; D015925:Cryopreservation; D005455:Fluorescent Antibody Technique; D006801:Humans; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D051379:Mice; D008810:Mice, Inbred C57BL; D008875:Middle Aged; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism*; D028361:Mitochondrial Diseases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D019636:Neurodegenerative Diseases / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism* / Q000648:ultrastructure; D013194:Staining and Labeling; D016707:Tissue Fixation / Q000379:methods*
chemical_list: D020136:Hydrogel, Polyethylene Glycol Dimethacrylate