Hi Tim, Well done
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 Morken, Timothy Sent: Thursday, 31 January 2013 6:09 AM To: Histonet Subject: [Histonet] ATPase without barbital buffer... info Well, I finally found the original papers on the subject of non-barbital buffers for ATPase from the Histonet posting below from Tony Henwood: The reference given for the procedure was : "Loughlin, M. (1993). Muscle biopsy. A laboratory investigation. Butterworth-Heinemann pp.78-79." But that is a book and one not available through our store. So I went online and was able to get the book for ....$2.00. yes, Two Dollars! Now I can give everyone the original references in the book. We have not tried this yet but plan to this year. The book actually has several ATPase methods that do not use barbital buffer on pages 77 - 82 (1993 edition). (One note. The page reference given by Tony's posting below seems to be the Brooke and Kaiser pH 4.3 and 4.6 method on page 81-82 combined with the Round pH 9.4 method on pp 80-81 1) Tris incubating buffer pH 9.4 (Louglin pp 77-79) a. Hayashi and Freiman, An Improved Method of Fixation for Formalin Sensitive Enzymes with Special Reference to Myosin Adenosine Triphosphatase, J Histochem & Cytochem, 1966 14: 577-581 2) Glycine incubating buffer pH 9.4 (Laughlin pp 80-81 a. Round, et. Al. Quick, Simple and Reliable Method for ATPase in Human Muscle Preparations. The Histochemical Journal, Nov 1980, Vol 12, Issue 6, pp 707-710 3) Acetate incubating buffer pH 4.3 and 4.6 ("reverse" ATPase) (Laughlin pp 81-82 a. Brooke, M.H. and Kaiser, K.K., Muscle Fibre Types: How many and what kind? Archives of Neurology, (Chicago), 1970, 23, 369-379 Another non-barbital method is: Khan, M.A., et al, A calcium-citro-phosphate technique for the histochemical localization of myosin ATPase, Stain Technology, 1972, Vol. 47, No. 6, pp. 277-281. Tim Morken Supervisor, Electron Microscopy/Neuromuscular Special Studies Department of Pathology UC San Francisco Medical Center ************************************************************ ATPase with sodium acetate buffer Tony Henwood AnthonyH <@t> chw.edu.au Sun Aug 20 18:14:40 CDT 2006 Here is our method using acetate buffer in place of barbiturate buffer: Adenosine Triphosphatase (ATPases) [Note: this is the Brooke and Kaiser 4.3 and 4.6 method combined with the Round 9.4 method. tm] Use Demonstration of muscle fibre types. Underlying Principle The principle relies on the ability of the enzyme to remove the terminal phosphate from the ATP, which then combines with calcium in the incubation solution to form an insoluble calcium phosphate. Cobalt is then exchanged for the calcium, which, after reaction with ammonium sulphide, forms a black, insoluble cobalt sulphide at the site of enzyme activity. Fixation and Sectioning Air dried unfixed 8µm cryostat sections Reagents 1. 1% calcium chloride a. Calcium chloride 5.0 g b. Distilled water 500ml 2. 2% Cobalt chloride Warning: Suspected Carcinogen - see MSDS a. Cobalt chloride 10.0 g b. Distilled water 500 ml 3. 1% Ammonium sulphide Warning: Flammable liquid, Irritant, Toxic stench - see MSDS a. 20% ammonium sulphide 0.5 ml b. Distilled water 9.5 ml 4. Acid Pre-incubation medium a. 0.2M Sodium Acetate i. Sodium Acetate Anhydrous 0.82 g ii. Distilled water 50 ml iii. b. 0.2M Acetic Acid i. Glacial Acetic Acid 0.6 ml ii. Distilled water 50 ml 5. 0.2M Acetate Buffer: pH 4.3 pH 4.6 a. 0.2M Sodium Acetate 11 ml 18 ml b. 0.2M Acetic Acid 12 ml 13 ml Adjust to pH 4.3 or 4.6 with 0.2M Sodium Acetate or 0.2M Acetic Acid ? 6. 7.5% Calcium Chloride a. Calcium Chloride 7.5g b. Distilled water 100ml 7. 7.05% Glycine a. Glycine 7.05g b. Distilled water 100ml Freeze in 2ml aliquots, defrost one aliquot before use. 8. 5.625% Sodium Chloride a. Sodium Chloride 5.625g b. Distilled water 100ml 9. 3.5% Sodim Hydroxide a. Sodium Hydroxide 3.5g b. Distilled water 100ml 10. Alkaline Stock Warning: Irritant - see MSDS a. 7.05% Glycine 2ml b. 7.5% Calcium Chloride 2ml c. 5.625% Sodium Chloride 2ml d. 3.5% Sodium Hydroxide 2ml e. Distilled water 42ml 11. Incubating Medium a. Alkaline Stock 25 ml b. ATP (Sigma A-7699) 40 mg Adjust to pH 9.4 - 9.5 with 0.1M HCl Method for preparing 9.4 Substrate Solution 1. Prepare fresh Alkaline Stock for each run 2. Place in 37oC Oven for 20minutes 3. pH to 11 using 0.1M NaOH (to activate the ATP) 4. Add ATP 5. pH to 9.4 using 0.1M HCl Staining Method 1. Place one slide in 4.3 and one in 4.6 buffer at room temperature for 20 minutes 2. Wash each in distilled water 3 times 3. Place these slides (one from the 4.3 solution , one from 4.6 solution and the 9.4 slide) in the 9.4 substrate at 37°C for: a. pH 9.4 10 mins b. pH 4.6 30 mins c. pH 4.3 45 mins 4. Place slides in 2 changes of 1% calcium chloride 3 min each 5. Place slides in 2% cobalt chloride 3 min 6. Wash well in distilled water 7. In fume cupboard drain slides well and place in ammonium sulphide solution for 1 min 8. Wash well in tap water 9. Dehydrate clear and mount. Results pH 9.4 Type 1 fibres pale Type 2A fibres intermediate Type 2B fibres dark pH 4.3 and pH 4.6 pH 4.3 pH 4.6 Type 1 fibres dark dark Type 2A fibres pale pale Type 2B fibres pale intermediate Type 2C fibres intermediate dark Notes This is a complicated stain and there are several areas in which one needs to be careful in order to achieve a good fibre type differentiation. 1. The pH of all solutions is critical 2. Timing is crucial a. The stock ammonium sulfide must still be yellow. As it ages or oxidizes, it becomes more red and cannot be used. 3. The pH of all solutions must be adjusted at the temperature they will be used. References 1. Loughlin, M. (1993). Muscle biopsy. A laboratory investigation. Butterworth-Heinemann pp.78-79. [actually pp 81-82. tm] Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) Laboratory Manager & Senior Scientist The Children's Hospital at Westmead, Locked Bag 4001, Westmead, 2145, AUSTRALIA. 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