Fluorometric examination of tissue amyloid fibrils in murine senile amyloidosis: use of the fluorescent indicator, thioflavine T.
Thioflavine T, a fluorescent indicator of amyloid fibrils (Naiki H, Higuchi K, Hosokawa M, Takeda T: Anal Biochem 177:244, 1989), has been tested in unfractionated tissue homogenates. With 250 nM thioflavine T, liver homogenates from a 17-month-old SAM-P/1 (senescence accelerated mouse-prone), which contained amyloid fibrils in murine senile amyloidosis (fASSAM) fluoresced brightly, whereas normal liver homogenates showed a negligible fluorescence. The following evidence confirmed that the fluorescence in the unfractionated preparations specifically represented fASSAM. The fluorescence of fASSAM deposited liver tissue was diminished to the level of the normal liver when the structure of the amyloid fibrils was disrupted by guanidine-HCl. Second, about 90% of the fluorescence of fASSAM deposited liver tissue was fractionated in the water-soluble fraction, in which amyloid fibrils are generally enriched. We then determined the concentration of fASSAM in the tissue, from the fluorescence and using the standard curve described in the above mentioned report. Lower limits of fASSAM determination were about 1 microgram/mg of tissue. A marked regional heterogeneity of fASSAM deposition was observed in the liver. We observed a linear correlation between fASSAM concentration and percentage of amyloid positive area in the liver. Age-dependent increases in fASSAM concentrations and total fASSAM contents were noted in the liver and spleen of 11 to 15-month-old SAM-P/1. There was also a positive correlation between the organ weight and fASSAM concentrations in both organs. Thus, thioflavine T is of practical use for the determination of fASSAM concentrations in the tissue.
Naiki
H
H
Department of Senescence Biology, Chest Disease Research Institute, Kyoto University, Japan.
Higuchi
K
K
Matsushima
K
K
Shimada
A
A
Chen
W H
WH
Hosokawa
M
M
Takeda
T
T
D000375:Aging; D000682:Amyloid / Q000378:metabolism; D000686:Amyloidosis / Q000175:diagnosis* / Q000378:metabolism; D000818:Animals; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D008099:Liver / Q000378:metabolism*; D051379:Mice; D008815:Mice, Inbred Strains; D013050:Spectrometry, Fluorescence; D013154:Spleen / Q000378:metabolism; D013844:Thiazoles
D000682:Amyloid; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D013844:Thiazoles; C009462:thioflavin T