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.