Amytracker Protocols
Staining of tissue sections
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.
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Antibody co-staining
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.
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Live-cell imaging
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.
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Fibrillation assays
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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In vivo staining
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.