Detection of cytochrome c oxidase activity and mitochondrial proteins in single cells.
Cytochrome c oxidase or mitochondrial respiratory chain complex IV is where over 90% of oxygen is consumed. The relationship between complex IV activity and mitochondrial proteins, which provides a guide to understanding the mechanisms in primary mitochondrial disorders, has been determined by histochemistry (activity) and immunohistochemistry in serial sections. In the central nervous system (CNS), mitochondrial activity and immunoreactivity have been determined in populations of cells in serial sections as capturing cells in more than one section is difficult. In this report we describe a method to determine complex IV activity in relation to mitochondrial proteins at a single cell level in the CNS. We performed complex IV histochemistry and immunohistochemistry consecutively in snap frozen sections. Although the product of complex IV histochemistry reduces the sensitivity of standard immunohistochemistry (secondary antibody and ABC method) the biotin-free Menapath polymer detection system (A. Menarini Diagnostics, Wokingham, UK) enables mitochondrial proteins to be detected following complex IV histochemistry. The co-occurring chromogens may then be separately visualised and analysed using multi-spectral imaging (Nuance system CRi, Woburn, MA). Our technique is applicable for exploring mitochondrial defects within single cells in a variety of CNS disorders and animal models of those diseases.
Mahad
D J
DJ
Mitochondrial Research Group, Institute for Ageing and Health, Newcastle University, Framlington Road, Newcastle upon Tyne NE2 4HH, UK. mahadd@doctors.org.uk
Ziabreva
I
I
Campbell
G
G
Laulund
F
F
Murphy
J L
JL
Reeve
A K
AK
Greaves
L
L
Smith
K J
KJ
Turnbull
D M
DM
D000328:Adult; D000368:Aged; D000818:Animals; D019069:Cell Respiration / Q000502:physiology; D002863:Chromogenic Compounds / Q000032:analysis / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000032:analysis* / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique / Q000379:methods; D006651:Histocytochemistry / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D008867:Microtomy / Q000379:methods; D028361:Mitochondrial Diseases / Q000175:diagnosis / Q000378:metabolism / Q000503:physiopathology; D024101:Mitochondrial Proteins / Q000032:analysis* / Q000378:metabolism; D009448:Neurochemistry / Q000379:methods*; D011108:Polymers; D011237:Predictive Value of Tests; D051381:Rats; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods
D002863:Chromogenic Compounds; D024101:Mitochondrial Proteins; D011108:Polymers; D003576:Electron Transport Complex IV