This article discusses the detection of amyloid deposits in tissue sections using hFTAA staining. Amyloids are associated with various proteopathic diseases and their visualization is crucial for understanding these conditions.
The deposition of misfolded protein aggregates - the amyloids in the extracellular tissue space are hallmarks of various proteopathic diseases. These amyloids are composed of beta sheets which align perpendicular to the fibrillar axis, forming cross beta-sheet repeats.
To detect the amyloid deposits with heptamer-formyl thiophene acetic acid, or hFTAA, stain, take a formalin-fixed, paraffin-embedded tissue section containing amyloid deposits.
Treat the sections with xylene to deparaffinize the tissue for improving the accessibility to stain in the subsequent steps. Add a few drops of the hFTAA staining solution over the tissue section and incubate to allow the dye to penetrate the tissue.
The hFTAA contains seven conjugated sulfur-containing thiophene units and terminal carboxyl groups attached to the thiophene backbone. These features provide flexibility to the unbound dye. During incubation, the end carboxyl groups of the dye bind with the positively charged amino acid residues lining the hydrophobic groove of the beta-sheet via electrostatic interactions.
Consequently, a conformational change occurs that reduces the structural flexibility of the bound hFTAA dye. This enhances the bound dye’s fluorescence quantum yield - the relative ratio of absorbed versus emitted photons. This process helps to improve the visualization of the amyloids.
Observe the slide under a fluorescence microscope. The presence of yellow-red fluorescence spots indicates the presence of amyloids in tissue sections.
Prepare the luminescent conjugated oligothiophene solution by resuspending lyophilized hFTAA in two millimolar sodium hydroxide to prepare a 1 mg/mL stock solution. Transfer the stock solution to a glass vial, and store at four degrees Celsius.
If formalin-fixed, paraffin-embedded, sections are used, de-paraffinize in xylene overnight. On the day of staining, immerse the sections in consecutive baths of 99% ethanol, 70% ethanol, dH2O, and PBS, for 10 minutes, each time. Then, allow the tissue sections to dry under ambient conditions.
While the tissue dries, prepare a working solution of hFTAA by diluting the stock 1:10,000 in PBS. When the tissue is dry, add droplets of the hFTAA working solution to each tissue section to cover it. Incubate for 30 minutes at room temperature.
Rinse off the staining solution with 500 microliters of PBS, and then, immerse the slide in the PBS bath for 10 minutes.
After allowing the section to dry under ambient conditions, mount using fluorescence mounting medium. Allow the mounting medium to settle overnight.
Amyloid detection can be performed directly after mounting, or even without mounting. However, if the goal of the experiment is to collect high-quality spectral information, overnight incubation is preferred.
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