Neuromuscular CLINICAL LABORATORY

ADENOSINE TRIPHOSPHATASE (ATP) STAINING PROTOCOL

PRINCIPLE:

The calcium method for ATPase demonstration, employing solutions of different pH values, have been used primarily to distinguish muscle fiber types. Muscle fibers may be broadly categorized as type 1 ("slow, red muscle, oxidative") and type 2 ("fast, white muscle, glycolytic"). Type 2 muscle fibers are further subdivided as 2a (glycolytic), 2b (glycolytic/oxidative), and 2c which we believe to be fibers that are changing types due to disease or injury. The way this stain is believed to work is as follows. The preincubation pH inactivates the myosin-ATPase enzyme of specific fiber types. The remaining active enzyme is attached to a calcium atom which is replaced by a cobalt and finally precipitated as a black insoluble compound by the ammonium sulfide.

QUALITY ASSURANCE:

This is a complicated stain and there are several areas in which one needs to be careful in order to achieve a good fiber type differentiation.

(1) The pH of all solutions is critical.

(2) Timing is crucial.

(3) Another source for inadequate differentiation is the pH solutions, particularly the sodium hydroxide, which should be not more than 2 weeks old (especially the 0.1 N).

(4) The stock ammonium sulfide must still be yellow. As it ages or oxidizes, it becomes more red and cannot be used.

SPECIMEN REQUIRED:

Snap frozen human striated muscle. (Use the isopentane freezing method described previously.)

METHOD

Fixation: None, use snap frozen tissue.

Technique: Cut 10 - 16 micron (12 µm) sections in cryostat from snap frozen biopsy. Attach one or more sections to a No.1½, 22 mm square coverslip.

Equipment:

ceramic staining rack - Thomas Scientific #8542-E40

columbia staining dish - Thomas Scientific #8542-C12

columbia staining dish(jar) - Thomas Scientific #8542-E30

forceps latex gloves

Reagents:

Adenosine triphosphate, disodium salt - Sigma A5394

Ammonium sulfide (light solution,original stock: concentration = 21 %) - Fisher A705-250, COMBUSTIBLE,ODORIFEROUS, USE IN HOOD

Calcium Chloride, anhydrous - Sigma C4901, store at room temperature

Canada Balsam, filtered neutral, Fisher B10-100

Cobalt chloride hexahydrate, ACS - Sigma C3169 - TOXIC, MUTAGENIC

Deionized water

Hydrochloric acid, ACS - Fisher A144-500, CORROSIVE, store at room temperature

Sodium acetate, trihydrate - Sigma S9513, store at room temperature

Reagent alcohol, ACS - histological Fisher A962-4 or HPLC A995, FLAMMABLE, TOXIC, TERATOGENIC, store at room temperature in flammable cabinet

Sodium barbital (5,5' dietyl barbituric acid) - Sigma B0500, NARCOTIC, TOXIC,CONTROLLED SUBSTANCE, store at room temperature

Sodium Hydroxide , Certified ACS pellets - Fisher S318, CAUTION CORROSIVE!!

Xylenes - Fisher #HC700-1GAL, FLAMMABLE, store room temperature in flammable cabinet

Solutions:

1. 0.1 M Sodium Barbital Solution (5.15 gm barbital powder (room temp.)+ deionized H2O 250 ml): store at room temperature

2. 0.18 M Calcium Chloride (2.65 g CaCl2·2H2O + deionized H2O 100 ml): store at room temperature

3. 1 w/v Calcium Chloride (5 g CaCl2·2H2O deionized H2O 500 ml): store at room temperature

4. 2 w/v Cobalt Chloride (4 g CoCl2·6H2O deionized H2O 200 ml): store at room temperature

5. Barbital Acetate Solution

Sodium barbital (sodium barbiturate) 1.47 g

Sodium acetate (NaC2H3O2·3H2O) 0.97 g

Deionized H2O: Final volume of 50 ml

6. 1 N Sodium Hydroxide

4 g NaOH + deionized H2O 100 ml

Store at room temperature

7. 0.1 N Sodium Hydroxide

1 ml 1 N NaOH + 9 ml deionized water

Store at room temperature

8. 1 N Hydrochloric Acid

10.4 ml concentrated (12 N) HCl added to deionized H2O 125 ml

Store at room temperature

9. 0.1 N Hydrochloric Acid

10 ml 1 N HCl to 90 ml deionized H2O

Store at room temperature

10. Pre-Incubating Solutions (prepare fresh for each stain)

A). "9.4 ATP": TYPE 2 FIBERS DARK, TYPE 1 FIBERS LIGHT, TYPE 2C FIBERS DARK INTERMEDIATE

4.0 ml 0.1 M Sodium Barbital

4.0 ml 0.18 M Calcium Chloride

12.0 ml deionized H2O

Adjust pH to 9.7 - 9.8 just prior to use with a few drops of 0.1 N NaOH (muscle other than human usually requires a pH of ~ 10.2)

B). "4.6 ATP": TYPE 1 FIBERS DARKEST, TYPE 2B&C FIBERS INTERMEDIATE, TYPE 2A LIGHTEST

5.0 ml Barbital Acetate Solution

10.0 ml 0.1 N HCl

4.0 ml deionized H2O

adjust pH between 4.60 4.62 just prior to use with a few drops of 1 N HCl (muscle other than human usually requires a pH of ~ 4.5)

C). "4.3 ATP": TYPE 1 FIBERS DARKEST, TYPE 2C FIBERS INTERMEDIATE, TYPE 2A&B LIGHTEST

5.0 ml Barbital Acetate Solution

10.0 ml 0.1 N HCl

4.0 ml deionized H2O

Adjust pH between 4.25 - 4.3 just prior to use with a few drops of 1 NHCl (muscle other than human usually requires a pH of ~ 4.2)

11. ATP Incubating Solution (volume here is sufficient for three (3) staining jars)

60 mg ATP powder (disodium salt, Sigma # A-5394)

6.0 ml 0.1 M Sodium Barbital

21.0 ml deionized H2O

3.0 ml 0.18 M Calcium Chloride

Add the calcium chloride last to prevent precipitation of ATP !

Prepare just prior to use and adjust pH to 9.4 (9.40 - 9.45) with a few drops of 1N NaOH

DO NOT ALLOW THE pH TO BECOME TOO ALKALINE (10.0) AS THIS WILL CAUSE THE ATP TO PRECIPITATE NECESSITATING STARTING OVER!

12. Alcohol 50 %

Reagent alcohol ~50 ml

Deionized H2O ~50 ml

13. Alcohol 70 %

Reagent alcohol ~70 ml

Deionized H2O ~30 ml

14. Alcohol 80 %

Reagent alcohol ~80 ml

Deionized H2O ~20 ml

15. Alcohol 95 %

Reagent alcohol ~95 ml

Deionized H2O ~5 ml

Staining Procedure

1. Place one coverslip for each biopsy in a separate, labeled columbia staining dish (Thomas Scientific #8542-C12) for each pre-incubating solution.

2. Incubate in the 4.6 and 4.3 solutions for exactly five (5) minutes at room temperature. The 9.4 solution should be added for fifteen (15) minutes at room temperature.

3. After the appropriate pre-incubation time periods, pour out the solution and rinse one time with deionized water.

4. Pour the ATP solution into the staining jar: 25 minutes for the 4.6 and 4.3 ATP stains and 15 minutes for the 9.4 stain.

5. Wash each staining jar with three (3) changes of 1% Calcium Chloride for a total of approximately ten (10) minutes.

6. Add 2% Cobalt Chloride to each jar for ten (10) minutes.

7. Wash with three (3) to five (5) changes of an approximately 1:20 solution of 0.1M Sodium Barbital (~ 5 ml 0. 1 M Sodium Barbital + deionized water ~ 100 ml).
Note: the initial wash should turn a faint blue in color.

8. Wash with five exchanges of deionized H2O.

9. THIS STEP SHOULD BE DONE IN A FUME HOOD!! NOXIOUS & TOXIC FUMES!!

A. Prepare 2 % v/v solution of ammonium sulfide (0.2 ml stock NH4SO2 + 9.8 ml D.I.H2O): 10 ml for each staining jar

B. Add this solution to each jar for 20 - 30 seconds (sections will appear very dark).

C. Rinse in the fume hood with approximately 5 changes of tap water.

10. Transfer the coverslips with the stained sections to a porcelain rack, cleaning the back side of the coverslip with a cotton tipped swab if necessary.

11. Dehydrate in ascending alcohols (50%,70%,80%,95% x 2, 100% x 2) in columbia staining dish (jar) - Thomas Scientific #8542-E30 and clear with at least two changes of xylene, also done in columbia staining dish (jar) - Thomas Scientific #8542-E30.

12. Mount coverslips onto labeled glass slides with CANADA BALSAM ONLY!

Results
PRE-INC pHTYPE 1 TYPE 2ATYPE 2BTYPE 2C
9.4light (0 +1)dark ( +3) dark (+3)dark (+2)
4.6dark ( +3)light ( 0 ) intermediate (+1 +2)intermediate (+1 +2)
4.3dark ( +3)light ( 0 ) light ( 0 )intermediate (+1 +2)

REFERENCES

1. Brooke MH, Kaiser, KK. Arch. Neurol., 23: 369 - 379, Oct. 1970.

2. Brooke MH, Kaiser KK. J. Histochem. Cytochem., 18: 670 - 672, 1970.

3. Dubowitz V, Brooke MH. MUSCLE BIOPSY: A MODERN APPROACH, W.B. Saunders Co., Ltd, London, 1973.

3. Sheehan and Hrapchak, HISTOTECHNOLOGY, 2nd Edition; Batelle Press, Columbus, 1987.

4. Planer GJ, Pestronk A, et. al., Muscle & Nerve 1992;15:258.

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7/3/2013