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.