Home, grave, Indice alfabetico, Collegamenti, Patologia, Molecole, Sindromi, Muscoli, Giunzioni neuromuscolari, Nervi, Spinale, Atassia, Anticorpi e Biopsia, Informazioni per i pazienti TRADUZIONE di Natale Marzari

PRINCIPLE:

The calcium method for ATPase demonstration, employing solutions of differenti pH values, hanno been used primarily to distinguish fibre muscolari types. Fibre muscolari may be broadly categorized as tipo 1 ("slow, red muscoli, oxidative") e tipo 2 ("fast, white muscoli, glycolytic"). Tipo 2 Fibre muscolari sono further subdivided as 2a (glycolytic), 2b (glycolytic/oxidative), e 2c which we believe to be fibre that sono changing types due to disease o lesione. The way this Colorazione 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 e finally precipitated as a black insoluble compound by the ammonium sulfide.

QUALITY ASSURANCE:

This is a complicated Colorazione e there sono numerosi sonoas in which one needs to be careful in order to achieve a good fiber tipo differenziazione.

(1) The pH of all solutions is critical.

(2) Tempi is crucial.

(3) Another source for inadequate differenziazione is the pH solutions, particularly the sodium hydroxide, which should be not più than 2 settimane old (specialmente the 0.1 N).

(4) The stock ammonium sulfide must still be yellow. As it ages o oxidizes, it becomes più red e cannot be used.

SPECIMEN REQUIRED:

Snap frozen human striated muscoli. (Use the isopentane freezing method described precedentemente.)

METHOD

Fixation: None, use snap frozen tissue.

Technique: Cut 10 - 16 micron (12 µm) sections in cryostat from snap frozen Biopsia. Attach one o più sections to a No.1½, 22 mm squsono coverslip.

Equipment:

ceramic Colorazioneing rack - Thomas Scientific #8542-E40

columbia Colorazioneing dish - Thomas Scientific #8542-C12

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

forceps latex gloves

Reagents:

Adenosina trifosfato, disodium salt - Sigma A5394

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

Calcio 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 o HPLC A995, FLAMMABLE, TOXIC, TERATOGENIC, store at room temperature in flammable cabinet

Sodium barbital (5,5' dietyl barbituric acid) - Sigma B0500, NARCOTIC, TOXIC,CONTROLLED SOSTANZA, 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 Calcio Chloride (2.65 g CaCl₂^.2H₂O + deionized water 100 ml): store at room temperature

3. 1 w/v Calcio Chloride (5 g CaCl_22H2O deionized water 500 ml): store at room temperature

4. 2 w/v Cobalt Chloride (4 g CaCl_26H2O deionized water 200 ml): store at room temperature

5. Barbital Acetate Solution

Sodium barbital (sodium barbiturate) 1.47 g

Sodium acetate (NaC₂H₃O_23H2O) 0.97 g

Deionized water: Final volume of 50 ml

6. 1 N Sodium Hydroxide

4 g NaOH + deionized water 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 water 125 ml

Store at room temperature

9. 0.1 N Hydrochloric Acid

10 ml 1 N HCl to 90 ml deionized water

Store at room temperature

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

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

4.0 ml 0.1 M Sodium Barbital

4.0 ml 0.18 M Calcio Chloride

12.0 ml deionized water

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

B). "4.6 ATP": TYPE 1 FIBERS DARKEST, TYPE 2b FIBERS INTERMEDIATE, TYPE 2a LIGHTEST

5.0 ml Barbital Acetate Solution

10.0 ml 0.1 N HCl

4.0 ml deionized water

adjust pH between 4.60 4.62 just prior to use con a few drops of 1 N HCl (muscoli 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 water

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

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

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

6.0 ml 0.1 M Sodium Barbital

21.0 ml deionized water

3.0 ml 0.18 M Calcio Chloride

Add the calcium chloride last to prevent precipitation of ATP !

Prepare just prior to use e adjust pH to 9.4 (9.40 - 9.45) con 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. Alcol 50 %

Reagent alcohol ~50 ml

Deionized water ~50 ml

13. Alcol 70 %

Reagent alcohol ~70 ml

Deionized water ~30 ml

 

14. Alcol 80 %

Reagent alcohol ~80 ml

Deionized water ~20 ml

 

15. Alcol 95 %

Reagent alcohol ~95 ml

Deionized water ~ 5 ml

 

Colorazioneing Procedure

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

2. Incubate in the 4.6 e 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. Dopo the appropriate pre-incubation time periods, pour out the solution e rinse one time con deionized water.

4. Pour the ATP solution into the Colorazioneing jar: 25 minutes for the 4.6 e 4.3 ATP colorazioni e 15 minutes for the 9.4 Colorazione.

5. Wash each Colorazioneing jar con three (3) changes of 1% Calcio Chloride for a total of approximately ten (10) minutes.

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

7. Wash con 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 con five exchanges of deionized H2O.

9. THIS STEP SHOULD BE DONE IN A FUME HOOD!! NOXIOUS e 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 Colorazioneing jar

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

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

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

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

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

Results

PRE-INC pH	TYPE 1	TYPE 2A	TYPE 2B	TYPE 2C
9.4	light (0 +1)	dark ( +3)	dark (+3)	dark (+3)
4.6	dark ( +3)	light ( 0 )	intermediate (+1 +2)	intermediate (+1 +2)
4.3	dark ( +3)	light ( 0 )	light ( 0 )	intermediate (+1 +2)

REFERENCES

1. Brooke, M.H. e Kaiser, K.K., Arch. Neurol., 23: 369 - 379, Oct. 1970.

2. Brooke, M.H. e Kaiser, K.K., J. Histochem. Cytochem., 18: 670 - 672, 1970.

3. Dubowitz, V. e Brooke, M.H. MUSCLE Biopsia: A MODERN APPROACH, W.B. Saunders Co., Ltd, London, 1973.

3. Sheehan e Hrapchak, HISTOTECHNOLOGY, 2a Edition; Batelle Press, Columbus, 1987.

4. Planer, G.J., Pestronk, A., et. al., Muscoli e Nervi, Feb. 1992.

PREPARED BY:	G. JAMES PLANER	10/22/90
SUPERCEDES REVISION	G. JAMES PLANER	06/20/95
REVIED BY	KAREN BIESER	07/18/97
ADOPTED	ALAN PESTRONK ,M.D.
REVIEWED:
REVIEWED:

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