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THIOFLAVIN S METHOD FOR AMYLOID IN
NEUROFIBRILLARY PLAQUES
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FIXATION: 10% buffered neutral formalin.
TECHNIQUE: Paraffin sections cut at 6-8 μm.
SOLUTIONS:
1% Thioflavin S
Thioflavin S, C.I. 49010 -------------------------------------------------------- - 1.0 gm
Distilled water -------------------------------------------------------------------- 100.0 ml
1% Thioflavin T
Thioflavin T, C.D. 49005 ---------------------------------------------------------- 1.0 gm
Distilled water -------------------------------------------------------------------- 100.0 ml
Modified Weigert's Iron Hematoxylin
(See Modified Puchtler's Congo Red Method)
0.5% Acid Alcohol Solution
(See Modified Puchter's Congo Red Method)
1% Acetic Acid
Acetic acid ----------------------------------------------------------------------- 1.0 ml
Distilled water -------------------------------------------------------------------- 100.0 ml
1. Deparaffinize and hydrate to distilled water.
2. Place in modified Weigert's iron hematoxylin for 15 seconds.
3. Rinse in three changes of distilled water.
4. Place in 0.5% acid alcohol for 5 seconds.
5. Rinse in four changes of distilled water.
6. Stain with 1% thioflavin S for 5 minutes. For all other tissues stain in 1% thioflavin T for 5 minutes.
7. Wash well in running water.
8. Place in 1% acetic acid for 15 minutes.
9. Rinse in three changes of distilled water.
10. Stand slide on end and thoroughly air dry.
11. Dip in xylene and mount with synthetic resin.
When thioflavin S is viewed with a fluorescence microscope amyloid in neurofibrillary plaques and in blood vessels will fluoresce intense yellow-white. Amyloid in tissues stained with thioflavin T will fluoresce an intense white.
Thioflavin S stains amyloid in neurofibrillary plaques in brain much better than thioflavin T. However, there is considerable non-specific background fluorescence. Thioflavin T stains amyloid well in other tissues and there is very little background fluorescence.
Most texts that contain the thioflavin S and thioflavin T methods use alum hematoxylin to quench background fluorescence. We found that Weigert's iron hematoxylin is more effective in quenching background fluorescence.
Schwartz, P.: Amyloid degeneration and tuberculosis in the aged. Gerontologia 18:321-362, 1972.
Vassar, P.S.and Culling, C.F.A.: Fluorescent stains, with special reference to amyloid and connective tissue. Arch. Pathol. 68:487, 1959.
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