RE: [Histonet] COX/SDH Combo staining
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 Bufferup 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 succinate12.96 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 Phosphate52ml 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 mitochondriaBrown. Cytochrome Oxidase negative mitochondriaBlue 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-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Helene Degan Sent: Wednesday, 10 July 2013 5:33 AM To: histonet@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
RE: [Histonet] COX/SDH Combo staining
Easier way to do this stain...since you do these two stains separately incubate the slide in a the COX solution for 1 hour rinse in distilled water and incubate in SDH solution for 1 hr. rinse 5min in physiological saline, 10min in 10% formalin saline, rinse in 15% ethanol then mount with Crystal mount media. Let dry then cover slip as usual Florence Leomiti HT (ASCP) Neuromuscular Lab Tech. Phone 503-494-6781 Fax 503-418-4249 Pager 16822 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tony Henwood (SCHN) Sent: Tuesday, July 09, 2013 4:18 PM To: 'Helene Degan'; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] COX/SDH Combo staining 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 Bufferup 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 succinate12.96 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 Phosphate52ml 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 mitochondriaBrown. Cytochrome Oxidase negative mitochondriaBlue 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
