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United States Government Work, uncopyrighted, public-domain,
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See also: Tissue Stains: 49,
51, 52,
56, 69,
72, 81.
PRINCIPLE OF THE TEST.
To stain paraffinized tissue sections
for reticulum, required in the diagnosis of certain diseases
encountered in anatomic pathology,
especially liver biopsies taken for Hepatitis C.
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.
Biebrich scarlet-acid Fuchsin solution.
Biebrich scarlet, aqueous, 1%.
Acid fuchsin, aqueous, 1%.
Glacial acetic acid.
Phosphomolybdic acid.
Phosphotungstic acid.
Aniline blue.
2% Light green solution.
1% Glacial acetic acid solution.
Weigert's iron hematoxylin solution.
STEP-BY-STEP DESCRIPTION.
FIXATION:
10% buffered neutral formalin
TECHNIQUE:
Cut paraffin sections at 6 microns
SOLUTIONS:
BIEBRICH SCARLET-ACID FUCHSIN SOLUTION
Biebrich scarlet, aqueous, 1%................... 90.0 ml
Acid fuchsin, aqueous, 1%....................... 10.0 ml
Glacial acetic acid.............................. 1.0 ml
PHOSPHOMOLYBDIC-PHOSPHOTUNGSTIC ACID SOLUTION
Phosphomolybdic acid............................. 5.0 gm
Phosphotungstic acid............................. 5.0 gm
Distilled water................................ 200.0 ml
ANILINE BLUE SOLUTION
Aniline blue..................................... 2.5 gn
Glacial acetic acid.............................. 2.0 ml
Distilled water................................ 100.0 ml
2% LIGHT GREEN SOLUTION.
1% GLACIAL ACETIC ACID SOLUTION.
Glacial acetic acid.............................. 1.0 ml
Distilled water................................ 100.0 ml
STAINING PROCEDURE.
1. Deparaffinize sections, and hydrate to distilled water.
2. Mordant overnight at room temperature if formalin fixed;
or for 1 hour at 56o C for Bouin's.
3. Cool and wash in running water until yellow color disappears.
4. Rinse in distilled water.
5. Weigert's iron hematoxylin solution for 10 minutes.
Wash in running water for 10 minutes.
6. Rinse in distilled water.
7. Biebrich scarlet-acid fuchsin solution for 2 minutes. Save solution)
8. Rinse in distilled Water.
9. Phosphomolybdic-phosphotungstic acid solution for 10 - 15 minutes
before aniline blue solution. (If using light green counterstain,
aqueous phosphotungstic acid, 5%, for 15 minutes.) Discard solution.
10. Aniline blue solution for 5 minutes (light green for 1 minute).
For central nervous system, stain 15 to 20 minutes. Save solutions.
11. Rinse in distilled water.
12. Glacial acetic solution for 3 to 5 minutes. Discard solution.
13. Dehydrate in 95% alcohol, absolute alcohol, and clear (two changes
each).
14. Mount.
CONTROL:
Skeletal muscle and skin.
RESULTS:
Nuclei................................................... black
Cytoplasm, keratin, muscle fibers, intercellular fibers... red
Collagen.................................................. blue
REFERENCES.
1. Jones ML.
Connective Tissues and Stains. Chapter 9, pp. 139-162.
In: Bancroft JD, Gamble M.
Theory and Practice of Histological Techniques. Fifth Edition.
Edinburgh: Churchill Livingstone. 2002;:139-162.
ISBN 0-443-06435-0, 796 pages.
2. Masson P.
Some histological methods.
Trichrome stains and their preliminary technique.
Bull Intl Assn Med. 1929;12:75.
3. 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.
4. 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.