Fluorescent Amyloid Stains

Fluorescent dye staining is a very sensitive technique for demonstrating various materials. This is, at least partly, due to the fact that the stained material glows on a darker background and therefore stands out as a light in the darkness. Although amyloid stained in this manner is not as bright as fluorescent staining of some other materials, it is still easier to see. The dye used is almost always thioflavine T, although thioflavine S has also been recommended.

Both thioflavine T and thioflavine S are mixtures. Thioflavine T is a basic dye with a yellow component which is the dye responsible for staining amyloid. Thioflavine S is an acid dye, belonging to the direct dye subclass (direct yellow 7). Keliényi thought that the components of thioflavine S may have differing numbers of thiazole rings, and that this influenced the way it stains. He points to the fact that primuline contains two components which differ in this regard, and that thioflavine S is made from primuline. Since thioflavine T is a basic dye and thioflavine S is an acid dye, he suggested that different reactive groups on the amyloid may be responsible for the selective staining. The fact that one is a direct cotton dye also raises the possibility that hydrogen bonding is involved.

The binding of thioflavine T was investigated by Khurana et. al. who suggested that the positive charges of the dye were involved in micelle formation. A micelle is defined in the Merriam Webster dictionary as:–

  "MICELLE
  A unit of structure built up from polymeric molecules or ions: as
  a : an ordered region in a fiber (as of cellulose or rayon)
  b : a molecular aggregate that constitutes a colloidal particle "

These micelles caused increased fluorescence of thioflavine T, and were bound to amyloid fibrils "involving both ionic and hydrophobic interactions." The inference is that the increased fluorescence of amyloid with thioflavine T which causes it to be selectively brighter than the background is due to the increased fluorescence of the micelles which attach to it.

Thioflavine S has become quite popular for the demonstration of neurofibrillary tangles and senile plaques in Alzheimer's disease since a modification to the original technique was made. However, 30µ free floating sections may be necessary. Unlike thioflavine T, which stipulates differentiation with dilute acetic acid, the thioflavine S methods specify differentiation in ethanol.

Fluorescent Staining Methods
Vassar & Culling Thioflavine T
Burn's thioflavine T
Thioflavine S for amyloid
Thioflavine S for Alzheimer's

 

Reference
Keliényi, G., (1967),
On the histochemistry of azo group-free thiazole dyes.,
Journal of histochemistry and cytochemistry, v 15, page 172-180.

Khurana, R., Coleman, C., Ionescu-Zanetti, C., Carter, S., Krishna, V., Grover, R. & Singh, S., (2005),
Mechanism of thioflavin T binding to amyloid fibrils.,
Journal of structural biology, v 151, page 229-238.

The Internet Pathology Laboratory for Medical Education, (here)
Mercer University School of Medicine.,
Thioflavine S for amyloid
Thioflavine S for senile plaques.

 


 

 

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