Source: Nyström, S. et al., Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging. J. Vis. Exp. (2017).
This video describes a technique to stain amyloids in tissue sections using a luminescent-conjugated oligothiophene dye – heptamer-formyl thiophene acetic acid, or hFTAA – for fluorescence imaging. Amyloids are β-sheet-rich abnormal protein aggregates. Their detection in tissue samples is helpful in the diagnosis and treatment of amyloid-associated diseases or proteinopathies.
1. LCO Staining Solution
NOTE: If hFTAA is purchased from a commercial vendor, please follow the vendor's instructions instead of section 1.
2. Preparation of Tissue Samples
NOTE: Many tissue types can be imaged using hFTAA as an amyloid marker. See Figure 1 for examples. hFTAA is sensitive to aggregate conformation. The staining should hence preferably be performed on undisrupted aggregates with no epitope exposure. Optimal spectral quality is achieved if the tissue fixation is kept to a minimum. Hence fresh frozen material, gently fixed in ethanol at the time of staining, is preferred. However, it is possible to detect amyloid deposits also in tissue that has been fixed with e.g., formalin. hFTAA generally penetrates the tissue well. Select a specimen thickness that is compatible with the intended imaging technique.
Figure 1: Various tissue types and proteins aggregates showing h-FTAA staining of: (a) Mallory-Denk bodies consisting of keratin aggregates in a liver (counterstained with DAPI), (b) p62-positive r inclusions in sporadic inclusion-body myositis (s-IBM) muscle tissue, (c) Amyloid of islet amyloid polypeptide in human pancreas, (d) Amyloid of Immunoglobulin light chain in human intestine, (e) Sheep scrapie (prions) in mouse brain, (f) Chronic wasting disease (prions) in mouse brain, (g) Aβ plaques in APP23 mouse brain, (h) Aβ pathology in APP/PS1 mouse brain, and (i) Fat biopsy smears from diagnostic samples of transthyretin amyloid in human patients graded 1 – 4 according to standard Congo red (CR) scoring. Yellow areas show hFTAA stained amyloid deposits and blue is autofluorescence from adipose tissue. The scale bar length is specified in each panel.
Figure 2: Examples of co-staining with antibodies.
(a) Co-staining with anti-serum amyloid protein A (AA) immunofluorescence and hFTAA of human AA amyloid on the same section. Upper left: AA antibody emission 640 nm; upper right hFTAA at 488 nm; bottom panel overlay image showing colocalization in yellow. (b) Antibody staining and hFTAA fluorescence on consecutive sections. Top panel: AA antibody DAB staining, bottom panel: hFTAA imaged with a long pass emission filter. Scale bar: 100 µm.
The authors have nothing to disclose.
hFTAA/Amytracker545 | Ebba Biotech | ||
Dako fluorescene mounting medium | Agilent technologies | GM304 | |
LeicaDM6000 | Leica | ||
Lumen 200 | Prior | ||
Spectraview system | ASI spectral imaging |