Thioflavin T (Basic Yellow 1) (Synonyms: ThT)

This plan only provides a guide, please modify it to meet your specific needs.

1. Prepare dyeing solution

(1) Dissolve Thioflavin T in 70% ethanol to create a 5 mM stock solution;

Note: It is recommended that the stock solution be filtered and sterilized using a 0.22μm filter membrane. It should be aliquoted according to a single dosage and stored in the dark at -4°C or -20°C to avoid repeated freezing and thawing.

(2) Further dilute the stock solution in culture medium or other buffer to a working solution with a final concentration of 5 μM.

Note: Please adjust the concentration of the working fluid according to the actual situation and prepare it for use.

2. Cell staining

(1) Culture adherent cells on sterile coverslips;

(2) Remove the coverslip from the culture medium, suck out the excess culture medium, and place the coverslip in a humid environment;

(3) Use appropriate buffer to wash the coverslip 2-3 times;

(4) Fix cells in 4% paraformaldehyde and fix at room temperature for 5-20 minutes. After fixation, wash the coverslip 2-3 times with appropriate buffer;

(5) Use PBS containing 0.1% Triton

(6) Add about 100μL of dye working solution from one corner of the coverslip, shake gently to make the dye evenly cover all cells, and incubate at 37 °C in the dark for 5-30 minutes;

Note: The optimal incubation time is different for different experiments. Please explore by yourself according to the specific experimental needs.

(7) After the incubation, discard the dye working solution and use a suitable buffer to wash the coverslip 2 to 3 times.

3. Microscope detection: The maximum excitation wavelength and emission wavelength of Thioflavin T after binding to amyloid fibrils are 450/482nm respectively.

Precautions:

① Fluorescent dyes all have quenching problems. Please try to avoid light to slow down fluorescence quenching;

② For your safety and health, please wear a lab coat and disposable gloves.

References:

[1]. H LeVine 3^rd. Thioflavine T interaction with synthetic Alzheimer's disease beta-amyloid peptides: detection of amyloid aggregation in solution. 1993 Mar;2(3):404-10. doi: 10.1002/pro.5560020312.

[2]. Daniel R Beriault, Geoff H Werstuck. Detection and quantification of endoplasmic reticulum stress in living cells using the fluorescent compound, Thioflavin T. 2013 Oct;1833(10):2293-301. doi: 10.1016/j.bbamcr.2013.05.020. Epub 2013 Jun 5.