[Histonet] ATPase without barbital buffer... info

Morken, Timothy Timothy.Morken <@t> ucsfmedctr.org
Wed Jan 30 13:08:49 CST 2013

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]


               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


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
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.


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


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.


1.            Loughlin, M. (1993). Muscle biopsy. A laboratory investigation. Butterworth-Heinemann
pp.78-79. [actually pp 81-82. tm]


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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