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Amytracker 480 is our blue fluorescent optotracer molecule for labeling protein aggregates with repetitive arrangement of β-sheets. It labels Aβ plaques and neurofibrillary tangles in tissue sections with AD pathology and α-synuclein aggregates in tissue sections with PD pathology. Specifically, Amytracker 480 has been used to study amyloid formation during abnormal coagulation.[1]

As all our optotracers, Amytracker 480 is exceptionally photostable and fluorogenic. When bound to a target, Amytracker 480 can be imaged using fluorescence microscopy and spectral information can be acquired using a fluorescence spectrophotometer. Use recommended filter sets as well as excitation- and emission wavelengths according to Table 1.

Table 1: Excitation- and emission wavelengths as well as recommended filter sets.
Exmax Emmax Recommended filter-sets
Amytracker 480 420 nm 480 nm DAPI

Amytracker 480 is available as a 1 mg/ml solution in volumes ranging from 10 - 200 µL (see volumes and prices in the list below). As our standard solvent, we use ultrapure water. Please contact us for custom options.

DISCLAIMER: Amytracker are exceptionally stable and there is no direct requirement of handling or storing them in the dark or refrigerated. However, evaporation of aqueous solvent can occur upon opening/handling. Please make sure to close the container carefully, spin down liquid and use up small volumes quickly.

contact us for custom options.

Amytracker 520 is our green fluorescent optotracer molecule for labeling protein aggregates with repetitive arrangement of β-sheets. It labels Aβ plaques and neurofibrillary tangles in tissue sections with AD pathology and α-synuclein aggregates in tissue sections with PD pathology.

As all our optotracers, Amytracker 520 is exceptionally photostable and fluorogenic. When bound to a target, Amytracker 520 can be imaged using fluorescence microscopy and spectral information can be acquired using a fluorescence spectrophotometer. Use recommended filter sets as well as excitation- and emission wavelengths according to Table 1.

Table 1: Excitation- and emission wavelengths as well as recommended filter sets.
Exmax Emmax Recommended filter-sets
Amytracker 520 460 nm 520 nm FITC, GFP

Amytracker 520 is available as a 1 mg/ml solution in volumes ranging from 10 - 200 µL (see volumes and prices in the list below). As our standard solvent, we use ultrapure water. Please contact us for custom options.

DISCLAIMER: Amytracker are exceptionally stable and there is no direct requirement of handling or storing them in the dark or refrigerated. However, evaporation of aqueous solvent can occur upon opening/handling. Please make sure to close the container carefully, spin down liquid and use up small volumes quickly.

contact us for custom options.

Amytracker 540 is our yellow fluorescent optotracer molecule for labeling protein aggregates with repetitive arrangement of β-sheets. It labels Aβ plaques and neurofibrillary tangles in tissue sections with AD pathology and α-synuclein aggregates in tissue sections with PD pathology.

As all our optotracers, Amytracker 540 is exceptionally photostable and fluorogenic. When bound to a target, Amytracker 520 can be imaged using fluorescence microscopy and spectral information can be acquired using a fluorescence spectrophotometer. Use recommended filter sets as well as excitation- and emission wavelengths according to Table 1.

Table 1: Excitation- and emission wavelengths as well as recommended filter sets.
Exmax Emmax Recommended filter-sets
Amytracker 540 480 nm 540 nm FITC, GFP, YFP

Amytracker 540 is available as a 1 mg/ml solution in volumes ranging from 10 - 200 µL (see volumes and prices in the list below). As our standard solvent, we use ultrapure water. Please contact us for custom options.

DISCLAIMER: Amytracker are exceptionally stable and there is no direct requirement of handling or storing them in the dark or refrigerated. However, evaporation of aqueous solvent can occur upon opening/handling. Please make sure to close the container carefully, spin down liquid and use up small volumes quickly.

contact us for custom options.

Amytracker 630 is our orange fluorescent optotracer molecule for labeling protein aggregates with repetitive arrangement of β-sheets. It labels Aβ plaques and neurofibrillary tangles in tissue sections with AD pathology and α-synuclein aggregates in tissue sections with PD pathology.

As all our optotracers, Amytracker 630 is exceptionally photostable and fluorogenic. When bound to a target, Amytracker 630 can be imaged using fluorescence microscopy and spectral information can be acquired using a fluorescence spectrophotometer. Use recommended filter sets as well as excitation- and emission wavelengths according to Table 1.

Table 1: Excitation- and emission wavelengths as well as recommended filter sets.
Exmax Emmax Recommended filter-sets
Amytracker 630 520 nm 630 nm PI, Cy3, TxRed, mCherry, Cy3.5

Amytracker 630 is available as a 1 mg/ml solution in volumes ranging from 10 - 200 µL (see volumes and prices in the list below). As our standard solvent, we use ultrapure water. Please contact us for custom options.

DISCLAIMER: Amytracker are exceptionally stable and there is no direct requirement of handling or storing them in the dark or refrigerated. However, evaporation of aqueous solvent can occur upon opening/handling. Please make sure to close the container carefully, spin down liquid and use up small volumes quickly.

contact us for custom options.

Amytracker 680 is our red fluorescent optotracer molecule for labeling protein aggregates with repetitive arrangement of β-sheets. It labels Aβ plaques and neurofibrillary tangles in tissue sections with AD pathology and α-synuclein aggregates in tissue sections with PD pathology. Specifically, Amytracker 680 has been used to study amyloid formation during abnormal coagulation, Lewy body formation in a seeding based neuronal model, accumulation of misfolded proteins in the nucleolus and intracerebral formation of Aβ plaques using multiphoton microscopy.

As all our optotracers, Amytracker 680 is exceptionally photostable and fluorogenic. When bound to a target, Amytracker 680 can be imaged using fluorescence microscopy and spectral information can be acquired using a fluorescence spectrophotometer. Use recommended filter sets as well as excitation- and emission wavelengths according to Table 1.

Table 1: Excitation- and emission wavelengths as well as recommended filter sets.
Exmax Emmax Recommended filter-sets
Amytracker 680 530 nm 680 nm PI, mCherry, Cy3.5

Amytracker 680 is available as a 1 mg/ml solution in volumes ranging from 10 - 200 µL (see volumes and prices in the list below). As our standard solvent, we use ultrapure water. Please contact us for custom options.

DISCLAIMER: Amytracker are exceptionally stable and there is no direct requirement of handling or storing them in the dark or refrigerated. However, evaporation of aqueous solvent can occur upon opening/handling. Please make sure to close the container carefully, spin down liquid and use up small volumes quickly.

contact us for custom options.

Amytracker Mix&Try Kit is our recommended option for starting out with using Amytracker. It contains a sample volume (10 µL) of each Amytracker. All Amytrackers label Aβ plaques and neurofibrillary tangles in tissue sections with AD pathology and α-synuclein aggregates in tissue sections with PD pathology. The molecules are exceptionally photostable and fluorogenic. When bound to a target, the Amytracker molecules are imaged using fluorescence microscopy. Spectral information can be acquired using a fluorescence spectrophotometer.

The Amytracker molecules differ with regards to affinity, cellular uptake and excitation and emission wavelengths (see Table 1). Testing each molecule will allow you to determine which Amytracker is best suited for your experiment.

Table 1: Excitation- and emission wavelengths as well as recommended filter sets.
Exmax Emmax Recommended filter-sets
Amytracker 480 420 nm 480 nm DAPI
Amytracker 520 460 nm 520 nm FITC, GFP
Amytracker 540 480 nm 540 nm FITC, GFP, YFP
Amytracker 630 520 nm 630 nm PI, Cy3, TxRed, mCherry, Cy3.5
Amytracker 680 530 nm 680 nm PI, mCherry, Cy3.5

DISCLAIMER: Amytracker are exceptionally stable and there is no direct requirement of handling or storing them in the dark or refrigerated. However, evaporation of aqueous solvent can occur upon opening/handling. Please make sure to close the container carefully, spin down liquid and use up small volumes quickly.

contact us for custom options.

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, 100 µl, 150 µl or 200 µl (all delivered in 50 µl vials). Please, contact us to find out more about Amytracker for in vivo use.

Quick Facts

Amytracker fluorescent tracer molecules are:

  • provided as ex vivo variant with 1 mg/ml concentration in volumes of 50 µl, 100 µl, 150 µl and 200 µl and as a Mix&Try Kit with 5x10 µl
  • 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
  • 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.

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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.

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