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Amytracker™

General Information

Amytracker™ are fluorescent tracer molecules providing high-quality visualization of protein aggregates. 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. All Amytracker™ molecules are highly fluorescent only when they are bound to their target. This opens the possibility to perform fibrillation assays and spectrophotometric detection of amyloids in liquid samples. Besides being fast and easy to use, Amytracker™ are non-toxic and are readily taken-up when applied to cells in culture. Further, Amytracker™ are photo- and thermostable and allow for easy handling in any application. Our Amytracker™ ex vivo products will make your life easy when used for staining of tissue sections, live-cell imaging or fibrillation assays. Amytracker™ ex vivo products are available in aliquots of 50 µl or 100 µl. Our Amytracker™ in vivo products are prepared as 10X concentrated, sterile solutions and available in 50 µl aliquots. Amytracker™ in vivo products are suitable for staining of amyloids in live animal models.

Amytracker™ Mix&Try Kit is our Test Kit for Getting Started

Amytracker™ Mix&Try Kit contains 10 µl of each fluorencent tracer available in the Amytracker™ series. Amytracker™480 is excited between 405-458 nm and fluorescence emission occurs between 470–550 nm. Amytracker™520 is excited between 405-488 nm and emission is detected between 500-600 nm. Amytracker™630 is excited between 458-514 nm and emission is detected between 600-650 nm. Amytracker™680 is excited between 530-565 nm emission is detected between 600-800 nm. Using all these different options will allow you to select the best Amytracker™ for your experiment.

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

contact us for custom options.

Amytracker™480 is our blue fluorescent tracer molecule for amyloid staining.

Amytracker™480 fluorescence is readily visualized using standard microscopy equipment. Excitation is achieved using the 405 nm laser line, and emission can be detected at 480 nm using the standard DAPI or FITC filter sets. 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 50 µl and 100 µl aliquots and as in vivo variant in a 50 µl aliquot. Please contact us for customs options.

contact us for custom options.

Amytracker™520 is our green fluourescent tracer molecule for amyloid staining.

Amytracker™520 fluorescence is readily visualized using standard microscopy equipment. Excitation is achieved using the 458 or 488 nm laser lines, and emission can be detected using the standard FITC 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 50 µl and 100 µl aliquots and as in vivo variant in a 50 µl aliquot. Please contact us for customs options.
contact us for custom options.

Amytracker™630 is our orange fluorescent tracer molecule for amyloid staining.

Amytracker™630 fluorescence is readily visualized using standard microscopy equipment. Excitation is achieved using the 488 or 514 nm laser lines, and emission can be detected using standard TRITC or TxRed 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 50 µl and 100 µl aliquots and as in vivo variant in a 50 µl aliquot. Please contact us for customs options.

contact us for custom options.

Amytracker™680 is our red fluorescent tracer molecule for amyloid staining.

Amytracker™680 fluorescence is readily visualized using standard microscopy equipment. Excitation is achieved using the 561 nm laser line, and emission can be detected at 680 nm using the standard Cy5 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 50 µl and 100 µl aliquots and as in vivo variant in a 50 µl aliquot. Please contact us for customs options.

contact us for custom options.

Amytracker™

  • are provided as ex vivo variant in volumes of 50 µl and 100 µl, as in vivo variant with 50 µl and as Mix&Try Kit
  • are available for ex vivo and in vivo use
  • 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
  • are non-toxic
  • are readily taken up into cells
  • are photo- and thermostable

Applications

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.
  • Amytracker™ is a trademark belonging to Ebba Biotech AB

Download Info

Protocol I: Staining of amyloids 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 guard 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.
  • Mount tissue sections and seal the coverslip onto the slide to prevent drying.

Fluorescence Microscopy:

  • Amytracker™480: Excite at 405 nm (standard laser line) and detect emission using the DAPI or FITC filter sets. Optional: 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.
  • Amytracker™520: Excite at 458 or 488 nm (standard laser lines) and detect emission using a standard FITC filter set. Optional: 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.
  • Amytracker™630: Excite at 488 or 514 nm (standard laser lines) and detect emission using standard TRITC or TxRed filter set. Optional: 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.
  • Amytracker™680: Excite at 561 nm (standard laser line) and detect emission using a standard Cy5 filter set. Optional: 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.

Download Protocol

Protocol II: Staining of amyloids 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.
  • Mount tissue sections and seal the coverslip onto the slide to prevent drying.

Fluorescence Microscopy:

  • Amytracker™480: Excite at 405 nm (standard laser line) and detect emission using the DAPI or FITC filter sets. Optional: 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.
  • Amytracker™520: Excite at 458 or 488 nm (standard laser lines) and detect emission using a standard FITC filter set. Optional: 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.
  • Amytracker™630: Excite at 488 or 514 nm (standard laser lines) and detect emission using standard TRITC or TxRed filter set. Optional: 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.
  • Amytracker™680: Excite at 561 nm (standard laser line) and detect emission using a standard Cy5 filter set. Optional: 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.

Download Protocol

Protocol III: Live-cell imaging

All Amytracker™ fluorescent tracers permeate the cell membrane of living cells without permeabilization. We recommend washing, but if you have sensitive cells, you might consider to skip the washing step. Due to their low bakground fluorescence and minimal interference with biological autofluorescence, we recommend Amytracker™630 or Amytracker™680 for live-cell imaging. As Amytracker™ don't 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: Excite at 405 nm (standard laser line) and detect emission using the DAPI or FITC filter sets. Optional: 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.
  • Amytracker™520: Excite at 458 or 488 nm (standard laser lines) and detect emission using a standard FITC filter set. Optional: 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.
  • Amytracker™630: Excite at 488 or 514 nm (standard laser lines) and detect emission using standard TRITC or TxRed filter set. Optional: 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.
  • Amytracker™680: Excite at 561 nm (standard laser line) and detect emission using a standard Cy5 filter set. Optional: 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.

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™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 amyloids in vivo

Amytracker™ can be used for intravenous injections or intraperitoneal injection in small animals to stain amyloids 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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