This work was supported by the National Institute of Aging (AG04418), National Institute on Drug Abuse, National Institute of Health-Karolinska Institutet Graduate Partnerships Program, Karolinska Institutet, Swedish Research Council, Swedish Brain Power, and Swedish Brain Foundation. Many thanks to Mattias Karlen and Dr. Giuseppe Coppotelli for creative support with Figure 1 and 2, respectively; Karin Pernold for technical assistance; and Drs. Barry J. Hoffer, Lars Olson, and Nils-G&ouml;ran Larsson for much helpful advice and discussion.

1. Tissue preparation for cryosectioning
<ol>
	<li>Sacrifice the animal by either cervical dislocation or decapitation, in accordance with available ethical permit.</li>
	<li>Quickly collect tissues of interest (e.g. brain), and rapidly freeze on dry ice (tissues may require freezing in isopentane or propane chilled with liquid nitrogen to obtain optimal morphology). Store tissues in aluminum foil at -80 &deg;C until ready to section.</li>
	<li>Embed frozen tissue in preparation for cryosectioning.</li>
	...

<ol>
	<li>Larsson, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+mitochondrial+DNA+mutations+in+mammalian+aging.">Somatic mitochondrial DNA mutations in mammalian aging.</a> Annu. Rev. Biochem. 79, 683-706 (2010).</li><li>Cottrell, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+mitochondrial+DNA+mutations+in+disease+and+aging.">Role of mitochondrial DNA mutations in disease and aging.</a> Ann. NY Acad. Sci. 908, 199-207 (2000).</li><li>Harman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biologic+clock:+the+mitochondria.">The biologic clock: the mitochondria.</a> J. Am. Geriatr. Soc. 20, 145-147 (1972).</li><li>Wallace, D. 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Neurol. 67, 849-854 (2010).</li><li>Nonaka, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+pathology+in+cytochrome+c+oxidase+deficiency.">Muscle pathology in cytochrome c oxidase deficiency.</a> Acta. Neuropathol. 77, 152-160 (1988).</li><li>DiMauro, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+encephalomyopathies.">Mitochondrial encephalomyopathies.</a> Neurol. Clin. 8, 483-506 (1990).</li><li>Bonilla, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+morphological+approaches+to+the+study+of+mitochondrial+encephalomyopathies.">New morphological approaches to the study of mitochondrial encephalomyopathies.</a> Brain. Pathol. 2, 113-119 (1992).</li><li>Brierley, E. J., Johnson, M. A., Lightowlers, R. N., James, O. F., Turnbull, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+mitochondrial+DNA+mutations+in+human+aging:+implications+for+the+central+nervous+system+and+muscle.">Role of mitochondrial DNA mutations in human aging: implications for the central nervous system and muscle.</a> Ann. Neurol. 43, 217-223 (1998).</li><li>Borthwick, G. M., Johnson, M. A., Ince, P. G., Shaw, P. J., Turnbull, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+enzyme+activity+in+amyotrophic+lateral+sclerosis:+implications+for+the+role+of+mitochondria+in+neuronal+cell+death.">Mitochondrial enzyme activity in amyotrophic lateral sclerosis: implications for the role of mitochondria in neuronal cell death.</a> Ann. Neurol. 46, 787-790 (1999).</li><li>Gellerich, F. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+respiratory+rates+and+activities+of+respiratory+chain+complexes+correlate+linearly+with+heteroplasmy+of+deleted+mtDNA+without+threshold+and+independently+of+deletion+size.">Mitochondrial respiratory rates and activities of respiratory chain complexes correlate linearly with heteroplasmy of deleted mtDNA without threshold and independently of deletion size.</a> Biochim. Biophys. Acta. 1556, 41-52 (2002).</li><li>Larsson, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+transcription+factor+A+is+necessary+for+mtDNA+maintenance+and+embryogenesis+in+mice.">Mitochondrial transcription factor A is necessary for mtDNA maintenance and embryogenesis in mice.</a> Nat. Genet. 18, 231-236 (1998).</li><li>Ekstrand, M. 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No conflicts of interest declared.

The combined COX/SDH histochemical method enables the visualization of cells with mitochondrial dysfunction. This technique, with early studies dating back to 1968, remains popular, with many considering it the "gold standard" for identifying mitochondrial diseases in patients14,19,26,27. It is now frequently used to investigate mtDNA mutation-driven aging and aging-related disorders 12,13,18,20,21,24. The COX/SDH double-labeling method is often used in parallel with other techniques to identify spe...

<table border="1">
<tr>
<td>Name of the reagent</td>
<td>Company</td>
<td>Catalogue number</td>
<td>Comments (optional)</td>
</tr>
<tr>
<td>Dry Ice</td>
<td>AGA Gas AB</td>
<td>block form</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Isopentane (2-methylbutane)</td>
<td>Sigma-Aldrich</td>
<td>277258 
 CAS: 78-78-4</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Cyrostat embedding solution</td>
<td>Sakura Finetek</td>
<td>Tissue Tek 4583</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Cryostat</td>
<td>Microm</td>
<td>Microm Model HM 500M</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Slides</td>
<td>Thermo Scientific</td>
<td>Super Frost Plus 
 Menzel Gl&auml;ser 
 J1800AMWZ</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Cover glasses 
 Borosilicate glass</td>
<td>VWR International</td>
<td>16004-098</td>
<td>24 x 50 mm</td>
</tr>
<tr>
<td>Filter Paper</td>
<td>Munktell Filter AB</td>
<td>Quality: 1350 
 Article Number: 242 001</td>
<td>430 x 430 mm</td>
</tr>
<tr>
<td>3,3&prime;-diaminobenzidine tetrahydrochloride (DAB)</td>
<td>Sigma-Aldrich</td>
<td>Sigma Liquid Substrate System, D7304</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Cytochrome c (Type III, from equine heart)</td>
<td>Sigma-Aldrich</td>
<td>C2506 
 CAS: 9007-43-6</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Bovine catalase (from liver)</td>
<td>Sigma-Aldrich</td>
<td>C9322 
 CAS: 9001-05-2</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Nitroblue tetrazolium (NBT)</td>
<td>Sigma-Aldrich</td>
<td>N6876 
 CAS: 298-83-9</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Sodium succinate</td>
<td>Sigma-Aldrich</td>
<td>S2378 
 CAS: 6106-21-4</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Phenazine methosulfate (PMS)</td>
<td>Sigma-Aldrich</td>
<td>P9625 
 CAS: 299-11-6</td>
<td>PMS is light sensitive. Shield from light.</td>
</tr>
<tr>
<td>Sodium azide</td>
<td>Sigma-Aldrich</td>
<td>S8032 
 CAS: 26628-22-8</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Xylene</td>
<td>VWR International</td>
<td>EM-XX0060-4 </td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Entellan</td>
<td>VWR International</td>
<td>100503-870</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Malonate 
 (Malonic acid)</td>
<td>Sigma-Aldrich</td>
<td>M1296 
 CAS: 141-82-2</td>
<td>&nbsp;</td>
</tr>
</table>

visualization of mitochondrial respiratory function using cytochrome c oxidase / succinate dehydrogenase (cox/sdh) double-labeling histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Watch this Scientific Journal Video about Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry at JoVE.com

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

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36.4K vues.  Karolinska Institutet. Le cytochrome c oxydase / sodium déshydrogénase (COX / SDH) double étiquetage méthode permet la visualisation directe de l'ADN mitochondrial des déficits enzymatiques respiratoires dans des coupes de tissus congelés frais. C'est une technique simple histochimiques et est utile dans les enquêtes sur les maladies mitochondriales, le vieillissement et les troubles liés au vieillissement.