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

Summary

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.

Abstract

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.

Protocol

1. Tissue preparation for cryosectioning

1. Sacrifice the animal by either cervical dislocation or decapitation, in accordance with available ethical permit.
2. 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 °C until ready to section.
3. Embed frozen tissue in preparation for cryosectioning.
4. Collect 14-μm cryostat sections at -21 °C (may need to adjust temperature ± 1-2 °C). Thaw sections onto slides using heating block, and store slides w....

Discussion

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 patients^14,19,26,27. 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.......

Materials

List of materials used in this article

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