This article describes a method for detecting amyloid aggregates in tissue sections using the fluorescent dye hFTAA and fluorescence lifetime imaging microscopy (FLIM). The technique allows for the differentiation of compact and unstable amyloid structures based on fluorescence lifetime measurements.
Take a tissue section with amyloid aggregates representing abnormal protein deposits.
The tissue is stained with the fluorescent dye heptamer-formyl thiophene acetic acid or hFTAA, which binds specifically to beta-sheet structures of amyloid fibrils.
Image the slide using a confocal microscope equipped with a fluorescence lifetime imaging microscopy unit, which measures the fluorescence lifetime of the dye molecules.
Optimize the microscope settings for effective excitation of the dye molecules.
When laser light illuminates the tissue, hFTAA excites and fluoresces.
Stronger dye binding in compact structures results in longer lifetimes, while weaker dye binding in less compact structures leads to shorter lifetimes.
Later, the software generates a color-coded image of the aggregate.
Colors, such as red and yellow, appear in the periphery, indicating shorter fluorescence lifetimes that reflect less compact, unstable amyloid structures.
While colors like blue and green in the core represent longer fluorescence lifetimes, reflecting more compact and stable amyloid structures.
On the day of staining, immerse the sections in consecutive baths of 99% ethanol, 70% ethanol, dH_2O, 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 to 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.
Switch the microscope to FLIM mode, set the pinhole to 20, the excitation wavelength to 490 nanometers, and the laser intensity to 0.5%. Use pulsed lasers at 40 megahertz. In the FLIM software, set up photon counting over 550 nanometers. In the Display Parameters window, follow the photon counting until the max count is around 4,000 photon counts. Save the file and export it as SPC image.
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