CLINICAL LABORATORY
PRINCIPLE:
The complex naphthol, naphthol acid phosphate,
is hydrolyzed by acid phosphatases present in the tissue, and
napthol derivatives are therby produced. The naphthol derivatives
couple with the unstable diazonium salt, hexazonium pararosanilin,
and other dyes (rosaniline, magenta II & new fuchsine) to produce a red azo dye that marks the site of enzyme activity.
NOTE: Use of pure pararosaniline produces a pale red stain. Basic fuchsin, which contains pararosaniline and other red dyes, produces a darker, and more useful, result.
SPECIMEN REQUIRED:
Snap frozen human striated muscle. (Use the
isopentane freezing method described elsewhere.)
Controls:
Use prostate gland as a positive control.
For a negative control, eliminate the enzymatic substrate.
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:
Calcium Chloride, anhydrous - Sigma C4901, store at room temperature
Formaldehyde, 37 % - Fisher F79-500, POISON, CARCINOGEN, store at room temperature
Hydrochloric acid, ACS - Fisher A144-500, CORROSIVE, store at room temperature
1-Napthyl acid phosphate, disodium salt (C10H7O4PNa2) - Sigma N7255
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 acetate, trihydrate - Sigma S9513, store at room temperature
Sodium barbital (5,5' dietyl barbituric acid) - Sigma B0500, NARCOTIC, TOXIC,CONTROLLED SUBSTANCE, store at room temperature
Sodium nitrite certified crystalline - Fisher S347 - STRONG OXIDIZER, COMBUSTIBLE
Xylenes
- Fisher #HC700-1GAL, FLAMMABLE, store room temperature
in flammable cabinet)
I. Barbital Acetate Solution
Sodium barbital(sodium barbiturate) 1.47 g
Sodium acetate(NaC2H3O2.3H2O) 0.97 g
Deionized water -> final volume of 50
ml
II. Basic Fuchsin - HCl Solution
Basic Fuchsin 0.5 g
Deionized water 10.0 ml
Concentrated Hydrochloric Acid 2.0 ml
Dissolve dye in water, Add acid, Heat gently, Cool to room temp & Filter
Store at 4oC.
III. Sodium Nitrite, 4% (w/v)
Sodium Nitrite (NaNO2) 0.4 g
Deionized water 10.0 ml
IV. Baker's Solution (modified)
Calcium Chloride (anhydrous) CaCl2 0.3 g
Formaldehyde, 37% 3.0 ml
Deionized water -> final volume of 100 ml
V. Incubating Solution:
Sodium alpha napthyl acid phosphate 20 mg
Barbital Acetate Solution 5.0 ml
Deionized water 13.0 ml
VI. Indicator Solution:
Basic Fuchsin - HCl Solution 0.8 ml
Sodium Nitrite Solution, 4% 0.8 ml
Mix well, let stand for 2 minutes, then add to Incubating Solution
Adjust pH to between 5.0 and 6.0 with alkali
VII. Alcohol 50 %
Reagent alcohol ~50 ml
Deionized water ~50 ml
VIII. Alcohol 70 %
Reagent alcohol ~70 ml
Deionized water ~30 ml
IX. Alcohol 80 %
Reagent alcohol ~80 ml
Deionized water ~20 ml
X. Alcohol 95 %
Reagent alcohol ~95 ml
Deionized water ~ 5 ml
STAINING PROCEDURE:
1. Place coverslips with sections in Baker's
Solution in a Columbia staining dish (Thomas Scientific #8542-C12)
for 5 minutes at room temperature.
2. Wash with three exchanges of tap or deionized H2O.
3. Add incubation solution and stain for at least one (1) hour at room temperature in a dark place.
4. Wash with three exchanges of tap or deionized H2O.
5. Dehydrate (fairly rapidly) in ascending
alcohols (50%,70%,80%,95% x 2, 100% x 2) in Columbia staining
dish(jar)s - Thomas Scientific #8542-E30.
6. Clear with at least 2 changes of xylene
also in a columbia staining dish(jar) - Thomas Scientific #8542-E30
7. Mount with PERMOUNT or another synthetic
organic mounting medium.
Results:
A red azo dye indicates sites of acid phosphatase activity.
NOTE
For demonstrating tartrate resistent acid phosphatase add 28mg/10ml of sodium tartrate to the incubating solution.
Remember to use a control batch without sodium tartrate using test and control sections.
REFERENCES:
1. Barka, T., 1961. In: SELECTED HISTOCHEMICAL
AND HISTOPATHOLOGICAL METHODS, S.W. Thompson; Charles C. Thomas,
Springfield, IL, 1966.
2. Barka, T. and Anderson, P.J., 1962. In:
THEORY AND PRACTICE OF HISTOTECHNOLOGY,
Sheehan, D.C. and Hrapchak, B.B., 2nd Edition 1980; Battelle Memorial
Institute, Columbus, OH, 1987.
Return to Neuromuscular Home Page
Return to Muscle Biopsy Stains
Return to Neuromuscular Evaluations
12/14/2011