eoe sub articles

EoE Sub Articles

1. LCO Staining Solution
NOTE: If hFTAA is purchased from a commercial vendor, please follow the vendor&#39;s instructions instead of section 1.
<ol>
	<li>Resuspend the lyophilized hFTAA in 2 mM NaOH to prepare a stock solution of 1 mg/mL. Keep the stock in a glass vial at 4 &#176;C. The stock can be stored for one year.</li>
	<li>On the day of staining, prepare a working solution by diluting the stock 1:10,000 in Phosphate-buffered saline (PBS).</li>
</ol>
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.
<ol>
	<li>If formalin-fixed, paraffin-embedded sections are used, deparaffinized in xylene overnight. Dip the sections in consecutive baths of 99% ethanol, 70% ethanol, dH2O, and PBS, 10 min in each. Allow the tissue sections to dry under ambient conditions. Caution: Xylene is always handled in a chemical fume hood. Xylene and other organic solvents are harmful.
	<ol>
		<li>Thaw cryosections in room temperature. Fix the tissue sections in 10% formalin overnight and rehydrate by dipping them in consecutive baths of 99% ethanol, 70% ethanol, dH2O, and PBS, 10 min in each. Allow the tissue sections to dry under ambient conditions.</li>
	</ol>
	</li>
	<li>Add droplets of the hFTAA working solution (approximately 200 &#956;L) to the tissue sections to cover it. The droplet should stay in place by surface tension. Incubate for 30 min at room temperature for staining.</li>
	<li>Rinse off the staining solution with 500 &#956;L PBS using a pipette and then immerse the slide in the PBS bath for 10 min. Allow the section to dry under ambient conditions.</li>
	<li>Mount using the fluorescence mounting medium. Allow mounting medium to settle overnight. 
	NOTE: hFTAA staining can be performed in combination with other staining methods such as immunofluorescence, cell- or organelle-specific markers, etc. To perform co-staining, run your complete staining protocol of choice and add hFTAA staining at the end, starting from step 2.2. See Figure 2 for examples. For immunofluorescence, preferably select a secondary antibody that is excited at 640 nm or higher. In this wavelength range, hFTAA does not absorb and hence cannot be excited and will not fluoresce. This ensures that no bleed-through is seen between hFTAA and antibody.</li>
</ol>

<table><tbody><tr><td>hFTAA/Amytracker545&amp;nbsp;</td><td>Ebba Biotech</td><td></td><td></td></tr><tr><td>Dako fluorescene mounting medium</td><td>Agilent technologies&amp;nbsp;</td><td>GM304</td><td></td></tr><tr><td>LeicaDM6000&amp;nbsp;</td><td>Leica</td><td></td><td></td></tr><tr><td>Lumen 200&amp;nbsp;</td><td>Prior</td><td></td><td></td></tr><tr><td>Spectraview system</td><td>&amp;nbsp;ASI spectral imaging</td><td></td><td></td></tr></tbody></table>

hftaa staining of amyloid deposits: a technique to visualize amyloid fibrils using oligothiophene-based dye for fluorescence imaging in tissue sections

Source: Nystr&#246;m, S. et al., <a href="https://www.jove.com/v/56279">Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging.</a> 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 &#946;-sheet-rich abnormal protein aggregates. Their detection in tissue samples is helpful in the diagnosis and treatment of amyloid-associated diseases or proteinopathies.

<img alt="Figure 1" src="/files/ftp_upload/21045/21045_Figure1.jpg" /> 
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 ...

hFTAA Staining of Amyloid Deposits: A Technique to Visualize Amyloid Fibrils Using Oligothiophene-Based Dye for Fluorescence Imaging in Tissue Sections

Source: Nystr&#246;m, S. et al., Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and ...

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&#8217;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.

hftaa-staining-amyloid-deposits-technique-to-visualize-amyloid

Research

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Biological Techniques

Staining Techniques

Tissue staining

1.1K ビュー. 出典:Nyström, S. et al., Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging. J. Vis. Exp. (2017). このビデオでは、蛍光イメージングに発光結合オリゴチオフェン色素(ヘプタメルホルミルチオフェン酢酸、またはhFTAA)を使用して組織切片のアミロイドを染色する技術について説明しています。アミロイドは、βシートに富?...

ビデオ: アミロイド沈着物のhFTAA染色:組織切片の蛍光イメージングのためのオリゴチオフェンベースの色素を用いたアミロイド線維の可視化技術 - 概念

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These amyloid fibrils can sequester endogenous metastable proteins as well as components of the proteostasis network (PN) and thereby exacerbate protein misfolding in the cell. There are a limited number of tools available to assess the aggregation process of amyloid proteins within an animal. We present a protocol for fluorescence lifetime microscopy (FLIM) that allows monitoring as well as quantification of the amyloid fibrilization in specific cells, such as neurons, in a noninvasive manner and with the progression of aging and upon perturbation of the PN. FLIM is independent of the expression levels of the fluorophore and enables an analysis of the aggregation process without any further staining or bleaching. Fluorophores are quenched when they are in close vicinity of amyloid structures, which results in a decrease of the fluorescence lifetime. The quenching directly correlates with the aggregation of the amyloid protein. FLIM is a versatile technique that can be applied to compare the fibrilization process of different amyloid proteins, environmental stimuli, or genetic backgrounds in vivo in a non-invasive manner.

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Fluorescence lifetime imaging monitors, quantifies and distinguishes the aggregation tendencies of proteins in living, aging, and stressed C. elegans disease models.

蛍光生涯イメージングを用いたエージングC.エレガンスにおけるアミロイド構造の特徴

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These ...

JoVE Journal

生化学

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process.
However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion.
Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Here, a method is presented for the analysis of protein aggregation kinetics in the nematode Caenorhabditis elegans. Animals from an age-synchronized population are imaged at different time points, followed by semiautomated inclusion counting in CellProfiler and fitting to a mathematical model in AmyloFit.

Caenorhabditisエレガンスにおける自動包接計数を用いた生体内のタンパク質凝集動態のモニタリング

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

検出細胞毒性アミロイド次感染症の肺による内皮細胞緑膿菌法

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

hFTAA Staining of Amyloid Deposits: A Technique to Visualize Amyloid Fibrils Using Oligothiophene-Based Dye for Fluorescence Imaging in Tissue Sections

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hFTAA Staining of Amyloid Deposits: A Technique to Visualize Amyloid Fibrils Using Oligothiophene-Based Dye for Fluorescence Imaging in Tissue Sections

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