TAB-PAINT imaging of alpha-synuclein fibrils using Nile Red

Prepare alpha-synuclein fibrils by diluting alpha-synuclein monomer 70micromolar (µM) in PBS (with 0.01% NaN₃) and incubating at 37°C in a shaker (200 rpm) for over 24h 0m 0s. You can store the fibrils at 4°C for later use.

Argon plasma clean cover glass (VWR collection, 631-0124) for 0h 30m 0s in a plasma cleaner (Expanded Plasma Cleaner, PDC-002, Harrick Plasma).

In the meantime:

• Filter phosphate-buffered saline (PBS) using a 0.02 μm syringe filter (6809-1102, Whatman).
• Dilute Nile Red in filtered PBS to a concentration of 1nanomolar (nM)
  Note

  If you purchase Nile Red in solid form, we recommend preparing a 1 mM solution of Nile Red in DMSO and freezing small aliquots for later use. For each experiment, always use a new aliquot of Nile Red to prepare the 1 nM dilution, as the dye doesn't store well at low concentrations.

Create a PLL-coated sample well on the cover glass:

Create a sample well on the cleaned cover glass by sticking a frame-seal slide chamber (9x9 mm, SLF0201, Bio-rad) on the glass.

Pipet 70µL of 0.01% PLL (0.01% poly-L-lysine solution, P4707, Sigma-Aldrich) into the well and wait for 0h 15m 0s. The PLL will coat the surface of the cover glass.

Use a pipet to remove the excess PLL from the well and immediately replace it with 70µL of filtered PBS.

Use a pipet to remove the excess filtered PBS from the well and replace with70µL filtered PBS. Gently pipet up and down in the corners of the well. Repeat this step 2 more times.

Add fluorescent beads for lateral drift correction:

Remove the excess PBS from the well using a pipet.

Using a pipet, add 50µL of 0.1 µm diameter TetraSpeck Microspheres (0.1 µm, fluorescent blue/green/orange/dark red, T7279, Invitrogen) into the well.

Gently pipet up and down a few times and wait for 0h 5m 0s.

Use a pipet to remove the excess solution from the well and replace with70µL filtered PBS. Gently pipet up and down in the corners of the well. Repeat this step 2 more times.

Use a pipet to remove the excess PBS from the well, add 50µL of alpha-synuclein fibrils and pipet up and down a few times in each corner of the sample well and wait for 0h 5m 0s

Remove excess solution using a pipet and add 50µL of filtered PBS to the well.

Remove excess solution again and replace by 50µL of 1nanomolar (nM) Nile Red.

Image the sample straight away and make sure it doesn't dry out during imaging.