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Amytracker

Amytracker Mix&Try is our Test Kit for Getting Started

Amytracker Mix&Try contains 10 µl of each fluorescent tracer molecule available in the Amytracker series. Using all these different options will allow you to select the best Amytracker for your experiment.

Following fluorescent tracer molecules are included in the Amytracker Mix&Try Kit:

  • Amytracker 480 (Excitation: 405-458 nm, Emission: 470–550 nm, Emax = 480 nm)
  • Amytracker 520 (Excitation: 405-488 nm, Emission: 500-600 nm, Emax = 520 nm)
  • Amytracker 540 (Excitation: 430-500 nm, Emission: 530-600 nm, Emax = 540 nm)
  • Amytracker 630 (Excitation: 458-514 nm, Emission: 600-650 nm, Emax = 630 nm)
  • Amytracker 680 (Excitation: 530-565 nm, Emission: 600-800 nm, Emax = 680 nm)

 

Amytracker Mix&Try is available as ex vivo variant. Please contact us for custom options.

contact us for custom options.

Amytracker 680 is our red fluorescent tracer molecule for staining of amyloids and other protein aggregates.

The fluorescence of Amytracker 680 is visualised using standard fluorescence microscopy equipment (confocal microscope, spectroscope or spectrophotometer). Excitation is achieved using the 561 nm laser line, and emission can be detected at 680 nm using the standard PI, mCherry or Cy3.5 filter set. The optical spectrum of Amytracker 680 also allows custom settings to be applied, using an excitation range of 530-565 nm and a detection range of 600-800 nm. With exceptionally high signal-to-noise ratio and spectral properties that are clearly distinguishable from biological autofluorescence, we recommend Amytracker 680 for fibrillation assays, live-cell imaging and in vivo studies.

Amytracker 680 is available as ex vivo variant in 1x 50 µl and 2x 50 µl, with a volume discount. Please contact us for custom options and to request a quote for our in vivo products.

contact us for custom options.

Amytracker 630 is our orange fluorescent tracer molecule for staining of amyloids and other protein aggregates.

The fluorescence of Amytracker 630 is visualised using standard fluorescence microscopy equipment (confocal microscope, spectroscope or spectrophotometer). Excitation is achieved using the 488 or 514 nm laser lines, and emission can be detected using the standard PI, Cy3, TxRed, mCherry or Cy3.5 filter sets. The optical spectrum of Amytracker 630 also allows custom settings to be applied within an excitation range of 458-514 nm and a detection range of 600-650 nm. With exceptionally high signal-to-noise ratio and spectral properties that are clearly distinguishable from biological autofluorescence, we recommend Amytracker 630 for fibrillation assays, live-cell imaging and in vivo studies.

Amytracker 630 is available as ex vivo variant in 1x 50 µl and 2x 50 µl, with a volume discount. Please contact us for custom options and to request a quote for our in vivo products.

contact us for custom options.

Amytracker 540 is our green/yellow fluorescent tracer molecule for staining of amyloids and other protein aggregates.

The fluorescence of Amytracker 540 is visualised using standard fluorescence microscopy equipment (confocal microscope, spectroscope or spectrophotometer). Excitation is achieved using the 440 nm laser line and emission can be detected at 540 nm using the standard FITC, GFP or YFP filter set. The optical spectrum of Amytracker 540 also allows custom settings to be applied, using an excitation range of 430-500 nm and a detection range of 530-600 nm. With excellent cellular uptake properties, we particularly recommend Amytracker 540 for live-cell imaging and in vivo studies.

Amytracker 540 is available as ex vivo variant in 1x 50 µl and 2x 50 µl volumes. Please contact us for custom options and to request a quote for our in vivo products.

contact us for custom options.

Amytracker 520 is our green fluorescent tracer molecule for staining of amyloids and other protein aggregates.

The fluorescence of Amytracker 520 is visualized using standard fluorescence microscopy equipment (confocal microscope, spectroscope or spectrophotometer). Excitation is achieved using the 458 or 488 nm laser lines, and emission can be detected using the standard FITC or GFP filter set. The optical spectrum of Amytracker 520 also allows custom settings to be applied within an excitation range of 405-488 nm and a detection range of 500-600 nm.

Amytracker 520 is available as ex vivo variant in 1x 50 µl and 2x 50 µl volumes. Please contact us for custom options and to request a quote for our in vivo products.

contact us for custom options.

Amytracker 480 is our blue fluorescent tracer molecule for staining of amyloids and other protein aggregates.

The fluorescence of Amytracker 480 is visualized using standard fluorescence microscopy equipment (confocal microscope, spectroscope or spectrophotometer). Excitation is achieved using the 405 nm laser line, and emission can be detected at 480 nm using the standard DAPI filter set. The optical spectrum of Amytracker 480 also allows custom settings to be applied within an excitation range of 405-458 nm and a detection range of 470-550 nm.

Amytracker 480 is available as ex vivo variant in 1x 50 µl and 2x 50 µl, with a volume discount. Please contact us for custom options and to request a quote for our in vivo products.

contact us for custom options.

General Information

Amytracker are fluorescent tracer molecules for high-quality visualization of protein aggregates.

The molecules become vividly colorful – highly fluorescent - when bound to a target in a sample. Targets are amyloids or protein aggregates containing amyloid fibrils and proto-fibrils. Therefore, using Amytracker, you can achieve reliable fluorescent staining of early, pre-fibrillar states of amyloids arising from a variety of amyloidogenic proteins or peptides in tissues from a wide range of species and perform fibrillation assays and spectrophotometric detection of protein aggregates in liquid samples.

Amytracker molecules are non-toxic and are readily taken-up when applied to cells in culture. Further, they are photo- and thermostable and allow for easy handling in any application. Our ex vivo products will simplify your life when performing staining of tissue sections, live-cell imaging or fibrillation assays. The ex vivo products are available in aliquots of 50 µl or 100 µl. The in vivo products are suitable for staining of protein aggregates in live animal models and custom volumes are prepared as 10X concentrated (10 mg/ml), sterile solutions.

Quick Facts

Amytracker fluorescent tracer molecules are:

  • provided as ex vivo variant with 1 mg/ml concentration in volumes of 50 µl and 100 µl and as a Mix&Try Kit with five variants of 10 µl
  • provided as in vivo variants in custom volumes with concentration 10 mg/ml
  • readily taken up into cells and tissues
  • ex vivo variants are diluted 1000-fold for staining of tissue sections and 500-fold for live-cell imaging and in vivo variants are diluted 2-fold
  • non-toxic
  • photo- and thermostable
  • fast
  • accurate

Applications

Quickstart Protocol

Amyloid staining in tissue sections (Protocol I)

  • use fixed- or fresh tissue
  • dilute Amytracker 1:1000 in PBS (pH 7.4)
  • apply generously to sample
  • incubate 5-30 min
  • wash with PBS (optional)
  • image at recommended excitation/emission

Live-cell imaging (Protocol III)

  • dilute Amytracker 1:500 in cell culture medium or PBS (pH 7.4)
  • apply generously to sample
  • incubate 30 min
  • wash with cell culture medium or PBS (optional)
  • image at recommended excitation/emission

Storage

  • Store Amytracker at 4°C.
  • Use the opened vial within 12 month.

Note

  • Amytracker is for research use only.
  • Amytracker is not for diagnostic use or use in humans.
  • Amytracker is not for resale.

Download Info

Protocol I: Staining of protein aggregates in tissue sections

Amytracker can be used to stain tissue sections prepared by the most common techniques like paraffin embedding and freezing. Formalin fixation works well for extracellular deposits, and fixation in ice-cold ethanol or acetone is recommended for best preservation of intracellular aggregates. Amytracker can be easily combined with your co-staining of choice as described in Protocol II: Antibody co-staining. As Amytracker are highly fluorescent only when bound to their target, washing steps might be omitted when working with sensitive tissues. It is not necessary to protect Amytracker from light, so incubations can be performed on the bench at room temperature. It is however crucial not to let Amytracker dry on the sample.

Solutions and Reagents:

Use Amytracker ex vivo variant, which is provided as concentrated solution. The following common reagents are required (not supplied):

  • ice-cold Ethanol, 95%
  • Phosphate buffered saline (PBS), pH 7.4
  • Deionized water
  • Mounting medium

Assay Procedure:

  • Fix tissue sections with method of choice. We recommend fixation with ice-cold ethanol (5 min) at room temperature.
  • Rehydrate tissue sections in a mix of ethanol and deionized water (1:1) for 5 min. The rehydration step may need to be repeated with lower ethanol ratio depending on the tissue.
  • Equilibrate sections in PBS for 5 min.
  • Dilute Amytracker in PBS 1:1000.
  • Apply diluted Amytracker generously. Use enough liquid to prevent the sections from drying out during incubation. Incubate for 30 min.
  • Wash 2 x 5 min in PBS (optional).
  • Mount tissue sections and seal the coverslip onto the slide to prevent drying.

Fluorescence Microscopy:

  • Amytracker 480: An excitation range of 405-458 nm and a detection range of 470–550 nm may be applied depending on available laser lines and filter sets. For imaging with Amytracker 480, we recommend using the standard DAPI filter set.
  • Amytracker 520: An excitation range of 405-488 nm and a detection range of 500-600 nm may be applied depending on available laser lines and filter sets. For imaging with Amytracker 520, we recommend using the standard FITC or GFP filter set.
  • Amytracker 540: An excitation range of 405-488 nm and a detection range of 500-600 nm may be applied depending on available laser lines and filter sets. For imaging with Amytracker 540, we recommend using the standard FITC, GFP or YFP filter set.
  • Amytracker 630: An excitation range of 458-514 nm and a detection range of 600-650 nm may be applied depending on available laser lines and filter sets. For imaging with Amytracker 630, we recommend using the standard PI, Cy3, TxRed, mCherry or Cy3.5 filter set.
  • Amytracker 680: An excitation range of 530-565 nm and a detection range of 600-800 nm may be applied depending on available laser lines and filter sets. For imaging with Amytracker 680, we recommend using the standard PI or Cy3.5 filter set.

Download Protocol

Protocol II: Staining of protein aggregates with Amytracker and antibody co-stain

Amytracker might be combined with antibody staining to confirm presence of specifc amyloidogenic proteins or peptides. This protocol describes a simple procedure of how to combine Amytracker with antibody staining using a sequence specific primary antibody and a fluorescently labeled secondary antibody. We assume you are using formalin-fixed, paraffing embedded tissues and that tissue sections were deparafinized and rehydrated prior to staining. Please make sure to select a secondary antibody with suitable fluorescent label (see below in section 'Fluorescence Microscopy'). We recommend a simple blocking/permeabilization procedure. Consider to adjust the blocking and permeabilization steps if necessary.

Solutions and Reagents:

Use Amytracker ex vivo variant, which is provided as 1000-fold concentrated solution. The following common reagents are required (not supplied):

  • Phosphate buffered saline (PBS), pH 7.4
  • Blocking/Permeabilization buffer, 4% BSA in PBS with 0.2% Triton-X
  • Primary antibody
  • Secondary antibody with fluorescent label
  • Mounting medium

Assay Procedure:

  • Block tissue sections in Blocking/Permeabilization buffer for 30 min at room temperature.
  • Incubate tissue sections with primary antibody diluted in Blocking/Permeabilization buffer at 4 °C over-night.
  • Rinse with Blocking/Permeabilization buffer.
  • Incubate tissue sections with secondary antibody diluted in Blocking/Permeabilization buffer at room temperature for 30 min.
  • Rinse with Blocking/Permeabilization buffer.
  • Wash 5 min in PBS.
  • Dilute Amytracker in PBS 1:1000.
  • Apply diluted Amytracker generously. Use enough liquid to prevent the sections from drying out during incubation. Incubate for 30 min.
  • Wash 2 x 5 min in PBS (optional).
  • Mount tissue sections and seal the coverslip onto the slide to prevent drying.

Fluorescence Microscopy:

  • Amytracker 480: An excitation range of 405-458 nm and a detection range of 470–550 nm may be applied depending on available laser lines and filter sets. We recommend using the standard DAPI filter set.
  • Amytracker 520: An excitation range of 405-488 nm and a detection range of 500-600 nm may be applied depending on available laser lines and filter sets. We recommend using the standard FITC or GFP filter set.
  • Amytracker 540: An excitation range of 405-488 nm and a detection range of 500-600 nm may be applied depending on available laser lines and filter sets. We recommend using the standard FITC, GFP or YFP filter set.
  • Amytracker 630: An excitation range of 458-514 nm and a detection range of 600-650 nm may be applied depending on available laser lines and filter sets. We recommend using the standard PI, Cy3, TxRed, mCherry or Cy3.5 filter set.
  • Amytracker 680: An excitation range of 530-565 nm and a detection range of 600-800 nm may be applied depending on available laser lines and filter sets. We recommend using the standard PI or Cy3.5 filter set.

Download Protocol

Protocol III: Live-cell imaging

All Amytracker fluorescent tracers cross the cell membrane of living cells without permeabilization. Of all Amytracker molecules, Amytracker 540 has the best uptake properties. We recommend washing, but if you have sensitive cells, you might consider to skip the washing step. Due to their low background fluorescence and minimal interference with biological autofluorescence, we recommend Amytracker 630 or Amytracker 680 together with Amytracker 540 for live-cell imaging. As Amytracker do not bleach easily, you can use them for confocal, time-lapse imaging or multiphoton microscopy. Use cell culture medium with or without serum according to the requirements of your cell culture.

Solutions and Reagents:

Use Amytracker ex vivo variant, which is provided as 500-fold concentrated solution. The following reagents are required (not supplied):

  • Cell culture medium

Assay Procedure:

  • Dilute Amytracker in cell culture medium 1:500.
  • Incubate your cells in Amytracker supplemented cell culture medium for 30 min under normal culture conditions.
  • Replace Amytracker supplemented medium with fresh cell culture medium.
  • Perform microscopy method of choice.

Fluorescence Microscopy:

  • Amytracker 480: An excitation range of 405-458 nm and a detection range of 470–550 nm may be applied depending on available laser lines and filter sets. For imaging with Amytracker 480, we recommend using the standard DAPI filter set.
  • Amytracker 520: An excitation range of 405-488 nm and a detection range of 500-600 nm may be applied depending on available laser lines and filter sets. For imaging with Amytracker 520, we recommend using the standard FITC or GFP filter set.
  • Amytracker 540: An excitation range of 405-488 nm and a detection range of 500-600 nm may be applied depending on available laser lines and filter sets. For imaging with Amytracker 540, we recommend using the standard FITC, GFP or YFP filter set.
  • Amytracker 630: An excitation range of 458-514 nm and a detection range of 600-650 nm may be applied depending on available laser lines and filter sets. For imaging with Amytracker 630, we recommend using the standard PI, Cy3, TxRed, mCherry or Cy3.5 filter set.
  • Amytracker 680: An excitation range of 530-565 nm and a detection range of 600-800 nm may be applied depending on available laser lines and filter sets. For imaging with Amytracker 680, we recommend using the standard PI or Cy3.5 filter set.

Download Protocol

Protocol IV: Fibrillation assay

This protocol describes how Amytracker can be utilized for fibrillation assays and detection of amyloids in liquid samples. As Amytracker molecules are highly fluorescent only when they are bound to their target, they are ideally suited for spectrophotometric analysis. We recommend to perform a titration to use Amytracker in the lowest concentration possible for your specific application. The experimental conditions used to induce protein misfolding and aggregation can vary considerably depending on the amyloidogenic protein or peptide. It is important to note that Amytracker fluorescence can vary depending on pH and ionic strength of the buffer. In this protocol, fibrillation of bovine insuline is performed in 2 M acetic acid and 0.5 M NaCl.

Solutions and Reagents:

Use Amytracker ex vivo variant, which is provided as concentrated solution. The following common reagents are required (not supplied):

  • Fibrillation solution: 2 M acetic acid and 0.5 M NaCl in deionized water
  • Bovine insulin
  • 96-well microtiter plate (round bottom)
  • Plate reader

Assay Procedure:

  • Prepare a dilution series of Amytracker (1:500, 1:1000, 1:5000, 1:10000, 1:50000) in Fibrillation solution and include a blank control.
  • Freshly prepare 10 mg/ml bovine insulin in Fibrillation solution.
  • Fill the wells of the microtiter plate with 50 µl Amytracker of each dilution as well as blank control.
  • Add 50 µl bovin insuline (10 mg/ml in Fibrillation solution) to each well.
  • Place microtiter plate into Plate reader and record emission every 10 min for 5 h. Note that excitation and emission are different for each Amytracker (see section 'Spectrophotometer Settings').

Spectrophotometer Settings:

  • Amytracker 480: Excite at 430 nm and collect emission at 480 nm. Optional: Record an emission spectrum (450 - 700 nm) with 430 nm excitation.
  • Amytracker 520: Excite at 470 nm and collect emission at 530 nm. Optional: Record an emission spectrum (490 - 700 nm) with 470 nm excitation.
  • Amytracker 540: Excite at 470 nm and collect emission at 540 nm. Optional: Record an emission spectrum (490 - 700 nm) with 470 nm excitation.
  • Amytracker 630: Excite at 510 nm and collect emission at 635 nm. Optional: Record an emission spectrum (530 - 800 nm) with 510 nm excitation.
  • Amytracker 680: Excite at 540 nm and collect emission at 680 nm. Optional: Record an emission spectrum (560 - 800 nm) with 540 nm excitation.

Download Protocol

Protocol V: Staining of protein aggregates in vivo

Amytracker can be used for intravenous injections or intraperitoneal injection in small animals to stain protein aggregates in vivo. It will readily cross the blood brain barrier and no acute toxicity of Amytracker has been observed. We recommend multiphoton microscopy as read-out.

Solutions and Reagents:

Use Amytracker in vivo variant, which is sterile and provided as 10 mg/ml solution in ultrapure water. The following common reagents are required (not supplied):

  • 2x Phosphate buffered saline (2xPBS), pH 7.4
  • Multiphoton microscope with tuneable laser

Assay Procedure:

  • Dilute Amytracker 1:1 in sterile 2xPBS to obtain a 5 mg/ml solution.
  • Inject diluted Amytracker (i.v or i.p.). Adjust the injected volume to a total amount of 5 mg/kg body weight.
  • You can expect to see staining of cerebral plaques as early as 6 min after injection.
  • The staining will last for prolonged periods of time.

Multiphoton Microscopy:

  • Amytracker 480: Excite at 800 nm using a tuneable laser and detect emission using.
  • Amytracker 520: Excite at 800 nm using a tuneable laser and detect emission using.
  • Amytracker 630: Excite at 800 nm using a tuneable laser and detect emission using.
  • Amytracker 680: Excite at 800 nm using a tuneable laser and detect emission using.

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