This article outlines a protocol for imaging amyloid-beta plaques in brain sections from a neurodegenerative mouse model. The method involves the use of specific antibodies and curcumin to visualize and confirm the presence of these plaques.
Start with brain sections from a neurodegenerative mouse model.
Incubate with a detergent-supplemented blocking solution to increase tissue permeability and block non-specific binding. Discard the solution.
Introduce primary antibodies targeting amyloid-beta plaques. Incubate with agitation for antibodies to interact with amyloid-beta plaques, then wash.
Add red fluorophore-tagged secondary antibodies and incubate in the dark to allow the antibodies to interact.
Wash to remove unbound antibodies, followed by an alcohol wash.
Add curcumin, which binds beta-sheet-rich amyloids with high affinity, then wash.
Next, treat with increasing alcohol concentrations, dehydrating the brain sections. Add xylene to remove any traces of reagents.
Mount a section on a slide and observe under a fluorescence microscope.
Use a 590-nanometer wavelength laser to excite the red fluorophores on the secondary antibodies. Capture the image.
Switch to a 480-nanometer wavelength laser to excite the curcumin, emitting green fluorescence.
Overlay the images; red and green fluorescence colocalization confirms amyloid-beta plaque in the brain tissue.
For labeling of cryostat sectioned specimens with anti-A beta antibodies, wash the samples three times with fresh PBS per wash in individual wells of a 12-well plate before blocking any nonspecific binding with 10% normal goat serum in PBS and 0.5% Triton x-100 for one hour at room temperature.
At the end of the incubation, discard the blocking solution, and incubate the samples with A beta specific antibody overnight at 4 degrees Celsius and 150 rotations per minute. The next morning, wash the sections with three 10-minute washes in fresh PBS per wash followed by incubation with an appropriate secondary antibody conjugated with a red fluorophore for one hour at room temperature protected from light.
At the end of the incubation, wash the sections three times with PBS followed by one wash with 70% alcohol. After the alcohol wash, incubate the sections with 10 micromolar curcumin for 10 minutes at room temperature followed by three, one minute 70% alcohol washes.
After the last wash, dehydrate the sections with 90% and 100% alcohol for one minute per concentration, and clear the sections two times for five minutes per immersion with fresh xylene per incubation. Then mount and image the sections as demonstrated.
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