This stain is used for the visual detection
of amyloid in muscle and nerve fresh frozen sections in patients
who have amyloidosis. The dyeing of amyloid is by a mechanism
similar to the direct textile dyeing of cotton. The linearity
of the dye configuration permits hydrogen bonding of the azo and
amine groups of the dye to similarly spaced carbohydrate hydroxyl
radicals of the amyloid substance. The use of a staining solution
containing high content of alcohol and free alkali releases native
internal hydrogen bonding between adjacent polysaccharide chains
and creates more potential sites for the binding of the dye.
A section of a biopsy with abundant
amyloid in the muscle is used as a positive control.
Snap frozen human striated muscle. (Use the
isopentane freezing method described elsewhere.)
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.
1. Place the coverslip with section in a ceramic
staining rack .
2. Immerse sections in Harris Hematoxylin for 30 seconds to one (1) minute.
3. Wash with tap water until the water is clear.
4. Filter the Congo Red solution into a columbia
staining dish (Thomas Scientific#8542-E40)
5. Place coverslips with sections in the columbia
staining dish with the filtered Congo Red solution for 3 minutes
at room temperature.
6. Wash with several exchanges of deionized
7. Dehydrate in ascending alcohol solutions
(50%, 70%, 80%, 95% x 2, 100% x 2) in columbia staining dish(jar)s
- Thomas Scientific #8542-E30.
8. Clear with xylene (3 - 4 x ) also inin columbia staining dish(jar)s - Thomas Scientific #8542-E30
9. Mount coverslip onto a labeled glass slide
with Permount or some other suitable organic mounting medium.
With the light microscope, amyloid deposits
are red to pink-red, nuclei are blue, other tissue elements are
largely unstained. Amyloid deposits show a "apple-green"
birefringence with the polarizing microscope.
1. Thompson, Samuel W. SELECTED HISTOCHEMICAL
AND HISTOPATHOLOGICAL METHODS, Charles C. Thomas, Springfield,
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,
3. Protocol received from: Neuromuscular Division, Johns Hopkins School of Medicine.