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United States Government Work, uncopyrighted, public-domain,
DRAFT COPY ONLY. This document does not necessarily represent the views
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made with the document.
PRINCIPLE OF THE TEST.
To stain paraffinized tissue sections for nerve dendrites, required in
the diagnosis of certain diseases encountered in anatomic pathology.
SPECIMEN REQUIRED.
All human tissue excised at surgery, outpatient clinics, and postmortems,
fresh or in fixative, along with a filled-out Tissue Examination Form
(U. S. Standard Form 515, USSF515).
REAGENTS, INSTRUMENTATION.
10% buffered neutral formalin.
Paraffin sections at 6 microns
2% Silver nitrate solution.
10% Silver nitrate solution.
Silver nitrate crystals.
Distilled water.
40% Sodium hydroxide solution.
Sodium hydroxide.
Ammoniacal silver solution.
20% Formalin solution.
Formalin, 37-40%.
Gold chloride solution.
Gold chloride, 1% aqueous.
5% Sodium thiosulfate solution.
95% alcohol.
Absolute alcohol.
Clearing.
Permount or Histoclad.
Control: brain slide.
STEP-BY-STEP DESCRIPTION.
FIXATlON: 10% buffered neutral formalin
TECHNIQUE: Cut paraffin sections at 6 microns
SOLUTIONS: 2% SILVER NITRATE SOLUTION
Silver nitrate................................ 2.0 gms
Distilled water.............................. 100.0 ml
10% SILVER NITRATE SOLUTION
Silver nitrate................................ 10.0 gm
Distilled water.............................. 100.0 ml
40% SODIUM HYDROXIDE SOLUTION
Sodium hydroxide.............................. 40.0 gm
Distilled water.............................. 100.0 ml
AMMONIACAL SILVER SOLUTION
Silver nitrate, 10%............................ 5.0 ml
Sodium hydroxide, 40%......................... 5 drops
Dissolve the resulting precipitate by adding concentrated
ammonium hydroxide drop by drop until the precipitate is
dissolved. Filter and dilute to 20 ml with distilled
water. Make fresh just before use.
20% FORMALIN SOLUTION
Formalin, 37-40%.............................. 20.0 ml
Tap water..................................... 80.0 ml
GOLD CHLORIDE SOLUTION
Gold chloride, 1% aqueous....................... 3 drops
Distilled water................................. 10 ml
5% SODIUM THIOSULFATE SOLUTION
STAINING PROCEDURE.
1. Deparaffinize and hydrate to distilled water.
2. 2% silver nitrate solution for 48 hours, in the dark.
3. Rinse quickly in double distilled water.
4. Ammoniacal silver solution 10 - 20 minutes,
or until sections turn a deep brown.
5. Rinse in distilled water.
6. Reduce in 20% formalin solution for five minutes.
Sections appear a dark brownish black.
7. Rinse thoroughly in distilled water.
8. Tone in gold chloride solution for one hour.
Sections will be a reddish violet color.
9. Rinse thoroughly in distilled water.
10. Sodium thiosulfate solution for 1 minute.
11. Wash in tap water.
12. Dehydrate in 95% alcohol, absolute alcohol and clear (two changes each).
13. Mount.
CONTROL: Brain
RESULTS:
Intracellular neurofibrils.................. black
Axis cylinders.............................. black
Dendrites................................... black
Background.................................. purplish
REFERENCES.
1. Chan K-K, Lowe J.
Techniques in Neuropathology. Chapter 18, pp. 371-414.
In: Bancroft JD, Gamble M.
Theory and Practice of Histological Techniques. Fifth Edition.
Edinburgh: Churchill Livingstone. 2002;:371-414.
ISBN 0-443-06435-0, 796 pages.
2. Bielschowsky M.
Die Silberimprägnation der Axenzylinder.
[German: Silver impregnation of axon cylinders].
Zentralbl f Neurol. 1902;21:579.
3. Cajal S, Ramón Y.
Contribución al conocimiento de la neuroglia del cerebro humano.
[Spanish: Contribution to the knowledge of the neuroglia
of the human cerebrum].
Travaux du Lab d Réch biolog d L'Univ. d Madrid. 1913;2:255.
4. Cajal S, Ramón Y.
Sobre un nuevo proceder de impregnación de la neuroglia y sus
resultados en los centros nerviosos de hombre y animales. [Spanish: Regarding
a new procedure for impregnation of neuroglia, and its results
in central nerves of man an animals].
Travaux du Lab d Réch biolog d L'Univ. d Madrid. 1913;2:219.
5. Holmes W.
Silver Staining of nerve axons in paraffin sections.
Anat Rec. 1943;86:157-187.
6. Holzer W.
Über eine neue Methode der Gliafasernfärbung.
[German: Regarding a new method of glial fiber staining].
Zentralbl f d gesamt Neurol u Psychiatr. 1921;69:354-360.
7. Zon L.
The physical chemistry of silver staining.
Stain Technol. 1936;11:53-67.
8. Coers C.
The vital wtaining of muscle biopsies with methylene blue.
J Neurol, Neurosurg, Psychiatry. 1952;15:.
9. Cox W.
Imprägnation des centralen Nervensystem mit Quecksilversalzen.
[German: Impregnation of the central nervous system with mercurial salts].
Arch Mikr Anat. 1891;37;16-21.
10. Davenport HA.
Acid versus neutral formalin as a neurological fixative.
Stain Technol. 1934;9:49-52.
11. Ehrlich P.
Über die Methylenblaureaction der lebenden Nervensubstancen.
[German: Regarding the methylene blue reaction in living nerve substances].
Dtsch med Wochenschr. 1886;12:49.
12. Einarson I.
A method for progressive selective staining of Nissl
and nucleus substance in nerve cells.
Am J Pathol. 1932;8:295.
13. Holmes W.
Silver Staining of nerve axons in paraffin sections.
Anat Rec. 1943;86:157-187.
14. Holzer W.
Über eine neue Methode der Gliafasernfärbung.
[German: Regarding a new method of glial fiber staining].
Zentralbl f d gesamt Neurol u Psychiatr. 1921;69:354-360.
15. Kluver H, Barrera A.
A method for the combined staining of cells
and fibers of the central nervous system.
J Neuropathol Exp Neurol. 1953;12:400.
16. Mallory FB.
Pathological Technique.
New York: Hatner. 1961;:.
17. Nauta WJH.
Über die sogenannte terminale Degeneration im Zentralnervensystem
und ihre Darstellung durch Silberimprägnation. [German: Regarding
so-called terminal degeneration in the central nervous system, and its
demonstration by silver impregnation.
Schweiz Arch Neurol Psychiatr. 1950;66:353-376.
18. Penfield W.
A method of staining oligodendroglia and microglia.
Am J Pathol. 1928;4:153.
19. Stern JB
Neue Silberimprägnationsversuche zur Darstellung der
Mikro- und Oligodendroglia
(An Celloidin-serieschnitten anwendbare Methode).
[German: New silver-impregnation experiments for the demonstration
of microglia and oligodendroglia
(methods applicable to celloidin serial sections)].
Zeitschr f d gesamt Neurol u Psychiatr. 1932;138:50.
20. Weigert C.
Zur markscheidenfärbung.
[German: Regarding myelin sheath staining].
Dtsch Med Wochenschr. 1891;:1184.
21. Zon L.
The physical chemistry of silver staining.
Stain Technol. 1936;11:53-67.
22. Wiedemann B, Kuhn C, Schwechheimer K, Waldherr R, et al.
Synaptophysin identified in metastases of neuroendocrine tumors
by immunocytochemistry and immunoblotting.
Am J Clin Pathol. 1988;87:560-569.
23. Weil A.
A rapid method for staining myelin sheaths.
Arch Neurol Pschyiatr. 1928;20:392.
24. Prophet EB, Mills B, Arrington JB, Sobin LH, eds.
Laboratory Methods in Histotechnology.
Washington, DC: Armed Forces Institute of Pathology. 1992;:.
ISBN 1-881041-00-X, 278 pages.
25. Mikel UV, ed.
Advanced Laboratory Methods in Histology and Pathology.
Washington, DC: Armed Forces Institute of Pathology. 1994;:.
ISBN 1-881041-13-1, 254 pages.