|
Giunzioni neuromuscolari, Nervi, Spinale, Atassia, Anticorpi e Biopsia, Informazioni per i pazienti File tradotto parzialmente. RICHIESTA DI TRADUZIONE a Natale Marzari |
 |
|
Dopo 41 anni e 5 mesi, nel maggio 2006 la
magistratura di Trento ha riconosciuto l'esistenza e la gravità di quella
malattia rara che nessuna altra istituzione o persona singola della provincia di
Trento ancora mi riconosce, e per negare la quale mi perseguita. Natale Marzari |
|
PRINCIPLE:
The complex naphthol, naphthol acid phosphate,
is hydrolyzed by acid phosphatases presenti in the tissue, e
napthol derivatives sono therby produced. The naphthol derivatives
couple con the unstable diazonium salt, hexazonium pararosanilin,
to produce a red azo dye to mark the site of enzyme activity.
SPECIMEN REQUIRED:
Snap frozen human striated muscoli. (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 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:
Calcio 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
α-napthyl acid phosphate, monosodium salt (C10H8O4PNa)- Sigma N7000
pararosaniline hydrochloride certified - Sigma P1528, store at room temperature
Permount - Fisher SP15-100, FLAMMABLE
HEALTH HAZARD
Reagent alcohol, ACS,- istochimiche Fisher A962-4 o 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 SOSTANZA, 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. Pararosaniline HCl Solution
Pararosaniline Hydrochloride (C.I. 42500) 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. e 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)
Calcio 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:
Pararosaniline 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 e 6.0 con alkali
VII. Alcol 50 %
reagent alcohol ~50 ml
deionized water ~50 ml
VIII. Alcol 70 %
reagent alcohol ~70 ml
deionized water ~30 ml
IX. Alcol 80 %
reagent alcohol ~80 ml
deionized water ~20 ml
X. Alcol 95 %
reagent alcohol ~95 ml
deionized water ~ 5 ml
Colorazioneing Procedure:
1. Place coverslips con sections in Baker's
Solution in a columbia Colorazioneing dish (Thomas Scientific #8542-C12)
for 5 minutes at room temperature.
2. Wash con three exchanges of tap o deionized
H2O.
3. Add incubation solution e Colorazione for at
least one (1) ore at room temperature in a dark place.
4. Wash con three exchanges of tap o deionized
H2O.
5. Dehydrate (fairly rapidly) in ascending
alcohols (50%,70%,80%,95% x 2, 100% x 2) in columbia Colorazioneing
dish(jar)s - Thomas Scientific #8542-E30.
6. Clear con at least 2 changes of xylene
also in a columbia Colorazioneing dish(jar) - Thomas Scientific #8542-E30
7. Mount con PERMOUNT o another synthetic
organic mounting medium.
Results:
A red azo dye indicates sites of acid phosphatase
activity.
REFERENCES:
1. Barka, T., 1961. In: SELECTED HISTOChimici
AND HISTOPATHOLOGICAL METHODS, S.W. Thompson; Charles C. Thomas,
Springfield, IL, 1966.
2. Barka, T. e Anderson, P.J., 1962. In:
THEORY AND PRACTICE OF HISTOTECHNOLOGY,
Sheehan, D.C. e Hrapchak, B.B., 2a Edition 1980; Battelle Memorial
Institute, Columbus, OH, 1987.
| PREPARED BY | G. JAMES PLANER | 07/22/85 |
| SUPERCEDES REVISION | G. JAMES PLANER | 04/27/94 |
| REVISED BY | KAREN BIESER | 07/18/97 |
| ADOPTED BY | ALAN PESTRONK, M.D. | |
| REVIEWED BY | ||
| REVIEWED BY | ||
| REPLACED |
Ritorno a Fonama.org Home Page
8/7/97
