Neuromuscular CLINICAL LABORATORY

α-NAPTHYL ACETATE PROTOCOL FOR ESTERASE STAINING

PRINCIPLE:

This protocol demonstrates the sites of non-specific esterases in tissue sections. This modification of the technique described by B. J. Davis is a simple one that demonstrates denervated muscle fibers and neuromuscular junctions

SPECIMEN REQUIRED

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

METHOD

Fixation: None, use snap frozen tissue

Technique: Cut 10 -16 micron (12 micron) sections in cryostat from snap frozen biopsy. Attach one or more sections to a No. 1 ½ 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

Filter paper (Baxter #f2217-070, Grade 363 Qualitative)

Reagents:

Acetone - Baxter #010-4 FLAMMABLE

α- naphthyl acetate - Sigma N8505 - store desiccated at -20oC

Deionized water

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

Basic Fuchsin - Santa Cruz 203731
(NOT: pararosaniline hydrochloride certified - Sigma P1528 or Fisher P389),
Store at room temperature

Permount - Fisher SP15-100, FLAMMABLE HEALTH HAZARD

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

Sodium nitrite certified crystalline - Fisher S347 or Sigma S2252, STRONG OXIDIZER, COMBUSTIBLE

Sodium dibasic phosphate (Na2HPO4) anhydrous, ACS (FW 141.96)-Sigma S9763 or Fisher S374, or Mallinckrodt 7917: Store at room temperature

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

Solutions:

1. 0.2 M Sodium Phosphate

Sodium phosphate dibasic, anhydrous (Na2HPO4) 5.678g

Deionized water 200 ml

Store at room temperature

2. 4% Pararosaniline HCl

Dissolve pararosaniline 0.5 g in deionized water 10 ml

Heat gently on a hot plate (DO NOT BOIL)

Add concentrated (12N) hydrochloric acid 2.5 ml

Cool to room temperature

Filter (Baxter #f2217-070, Grade 363 Qualitative)

Store refrigerated (0-5oC)

3. 4% Sodium Nitrite

Sodium nitrite (NaNO2) 0.5 g

Deionized water 12.5ml

Store refrigerated (0-5oC)

4. "Azotized Pararosaniline" (PREPARED FRESH FOR EACH STAIN)

4% Pararosaniline-HCl (Solution #2) 0.4 ml

4% Sodium nitrite (Solution #3) 0.4 ml

Allow to sit at room temperature for a few minutes. (Solution is amber in color.)

5. Staining Solution (PREPARED FRESH FOR EACH STAIN): Into a 30 ml glass beaker ADD IN THE ORDER STATED

α-naphthyl Acetate ~ 3 - 5mg

Acetone ~ 0.75ml

MIX WELL

Add 0.2 M sodium phosphate (Na2HPO4) 12.5m

(SOLUTION MAY BECOME CLOUDY - OK!)

MIX WELL

Add Solution #4 ("Azotized Pararosaniline") and MIX WELL

SOLUTION WILL CHANGE COLOR FROM YELLOW TO RED-BROWN IN LESS THAN FIVE (5) MINUTES !

6. Alcohol 50 %

Reagent alcohol ~50 ml

Deionized water ~50 ml

7. Alcohol 70 %

Reagent alcohol ~70 ml

Deionized water ~30 ml

8. Alcohol 80 %

Reagent alcohol ~80 ml

Deionized water ~20 ml

9. Alcohol 95 %

Reagent alcohol ~95 ml

Deionized water ~ 5 ml

Staining Procedure

1. Place coverslips into a columbia staining dish (Thomas Scientific #8542-012)

2. When the Staining Solution (Solution %) is gold to orange in color, add it to the coverslips in the staining dish for 5 minutes at room temperature.

3. Immediately place sections under running tap water for several minutes to wash the reaction product off the sections.

4. Clean back of coverslips with cotton swab.

5. Place coverslips with se4ctions in a ceramic rack (Thomas Scientific #8542-E40).

6. Dehydrate in ascending alcohol solutions (50%, 70%, 80%, 95% x 2, 100% x2) - in columbia staining dish(jar)s - Thomas Scientific #8542-E30.

7. Clear with xylene (x - 4x) also in columbia staining dish(jar)s - Thomas Scientific #8542-E30.

8. Mount coverslip onto a labeled glass slide with Permount or some other suitable organic mounting medium.

Results

Esterase activity is demonstrated in denervated fibers as a red-brown color with normal fibers exhibiting pale yellow to brown color (sometimes two fiber types may be distinguished.) Neuromuscular junctions, when present, are demonstrated by a dark red-brown deposit on the edge of a muscle fiber.

REFERENCES

1. Thompson, SELECTED HISTOCHEMICAL AND HISTOPATHOLOGICAL METHODS, Charles C. Thomas, Springfield, IL, 1966.


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8/12/97