[Histonet] COX/SDH Combo staining

[Tony Henwood (SCHN)]

Here is our technique:
Cytochrome Oxidase - SDH

Use

1.	Defects of cytochrome oxidase activity
2.	Demonstration of mitochondria

Underlying Principle

Combining the COX with the SDH can demonstrate COX negative fibres. These may be SDH positive and may be ragged red fibres. 3 of the 13 subunits of COX are encoded by mitochondrial DNA whereas SDH is encoded by nuclear DNA. Therefore SDH is not affected by mitochondrial DNA mutations. Ragged red fibres in mitochondrial myopathies are generally COX neg (except in MELAS). Intra fibre mosaicism (mixture of bluish COX deficient and brownish COX positive mitochondria within the same fibre are also well demonstrated by this combined technique).


Fixation and Sectioning

Air dried unfixed 8µM cryostat sections


Reagents

1.	PBS Buffer:

Dissolve one Dulbecco PBS Tablet in 100ml distilled water

2.	COX A Solution:
Warning: DAB is carcinogenic – Avoid contact with skin
Sucrose					0.75g
3,3' Diaminobenzidine.4HCl (DAB)	0.05g
Adjust to pH 7.6
PBS Buffer				up to 50ml

Aliquot in 2ml amounts (labelled CA), store at -20oC 

3.	COX B Solution:

      Catalase (Sigma C9322)			0.001g
                  Cytochrome C (Sigma C2037)		0.05g
                  Adjust to pH 7.6
	      PBS Buffer				up to 50ml
	                        
Aliquot in 2ml amounts (labelled CB), store at -20oC 

4.	Incubating medium

	Defrost one CA and one CB vial, mix and place in a 2-slide plastic slide mailer. Place mailer in a coplin jar for support.

5.	SDH Reagents 
	1	Stock 0.2M Sodium Dihydrogen Phosphate (NaH2PO4)  
		Sodium Dihydrogen Phosphate		23.996 g
		Distilled water					1000 ml
		Store at room temperature
		
	2	Stock 0.2M Disodium Hydrogen Phosphate (Na2HPO4)  
		Disodium Hydrogen Phosphate		28.392 g
		Distilled water					1000 ml
		Store at room temperature

	3	0.6M succinate solution (adjusted to pH 7.6)
		Warning: Irritant – see MSDS
		Sodium succinate				12.96 g
		Distilled water					64.00 ml
		1M HCl					0.40 ml
		pH to 7.6 with 0.2M Disodium Hydrogen Phosphate
		Distilled water				up to 80 ml
		Aliquot 800μl into eppendorf vials (labelled S) and store at -20C 


	3.	Yellow SDH Incubation Medium

		Stock 0.2M Sodium Hydrogen Phosphate	52ml
		Stock 0.2M Disodium Hydrogen Phosphate	348ml
		Nitro Blue Tetrazolium (NBT)		0.6 g
		Adjust to pH 7.6 with stock phosphate solutions
		Aliquot 4ml into tubes labelled “SY” and store at -20C (enough for 100 tubes)
		

	4.	Incubating medium (prepare fresh)

		1.	Defrost a vial of succinate solution (S) and a Yellow Incubation Solution (SY)
		2.	Add solution “S” to “SY”, prior to use, mix and place in a small plastic slide mailer.



Method
	
1.	Prepare incubation solution
2.	Immediately place frozen sectioned slides in incubation solution and incubate for two hours at room temperature.
3.	Check staining and replace for longer if required
4.	Rinse slides in distilled water and prepare SDH incubation medium.
5.	Add slides to SDH medium and incubate at 37oC for 1-2 hour.
6.	Rinse slides in distilled water.
7.	Rinse in water, dehydrate, clear and mount.

Results

Cytochrome Oxidase positive mitochondria 		Brown.
Cytochrome Oxidase negative mitochondria 		Blue
References

1.	Seligman etal (1968) J Cell Biol 38:1-14.
2. 	Loughlin M. (1993). Muscle biopsy. A laboratory investigation. Butterworth-Heinemann p.38-39.
3. 	Sheehan D, Hrapchak B. (1987). Histotechnology, 2nd Ed. Batelle Press, Columbus p306-307

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Laboratory Manager & Senior Scientist 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Helene Degan
Sent: Wednesday, 10 July 2013 5:33 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] COX/SDH Combo staining

Hi
I'm looking for COX/SDH combo staining procedures for muscle enzymes we currently do these two enzyme procedure separately and we our looking into combining them or any idea as to what sites I may find the information.
 
Thanks 
 
Helene
deganh <@t> upstate.edu
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