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
-20C
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 -20C (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: [email protected]
[mailto:[email protected]] On Behalf Of Helene Degan
Sent: Wednesday, 10 July 2013 5:33 AM
To: [email protected]
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
[email protected]
_______________________________________________
Histonet mailing list
[email protected]
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
*********************************************************************************
This email and any files transmitted with it are confidential and intended
solely for the use of the individual or entity to whom they are addressed. If
you are not the intended recipient, please delete it and notify the sender.
Views expressed in this message and any attachments are those of the individual
sender, and are not necessarily the views of The Sydney Children's Hospitals
Network.
This note also confirms that this email message has been virus scanned and
although no computer viruses were detected, The Sydney Childrens Hospital's
Network accepts no liability for any consequential damage resulting from email
containing computer viruses.
*********************************************************************************
_______________________________________________
Histonet mailing list
[email protected]
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
