[Histonet] ATPase without barbital buffer... info
Paula Sicurello
patpxs <@t> gmail.com
Wed Jan 30 14:43:00 CST 2013
Hi Tim,
Thanks for finding those protocols for us!
Perhaps we can all move away from using a controlled substance (what a
nightmare to purchase).
Paula
--
Paula Sicurello, HTL (ASCP)
Supervisor, Clinical Electron Microscopy Laboratory
Duke University Health System
Rm.#251M, Duke South, Green Zone
Durham, North Carolina 27710
P: 919.684.2091
On Wed, Jan 30, 2013 at 2:08 PM, Morken, Timothy
<Timothy.Morken <@t> ucsfmedctr.org> wrote:
> 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.
> Tel: 612 9845 3306
> Fax: 612 9845 3318
>
>
>
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