May 09, 2024

Public workspaceTAB-PAINT imaging of alpha-synuclein fibrils using Nile Red

DOI
dx.doi.org/10.17504/protocols.io.n2bvj31mwlk5/v1
  • 1University of Cambridge
Ezra Bruggeman
Open access
DOI: dx.doi.org/10.17504/protocols.io.n2bvj31mwlk5/v1
Protocol Citation: Ezra Bruggeman 2024. TAB-PAINT imaging of alpha-synuclein fibrils using Nile Red. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj31mwlk5/v1
Manuscript citation:
POLCAM: Instant molecular orientation microscopy for the life sciences Ezra Bruggeman, Oumeng Zhang, Lisa-Maria Needham, Markus Körbel, Sam Daly, Matthew Cheetham, Ruby Peters, Tingting Wu, Andrey S. Klymchenko, Simon J. Davis, Ewa K. Paluch, David Klenerman, Matthew D. Lew, Kevin O’Holleran, Steven F. Lee. bioRxiv 2023.02.07.527479, doi: https://doi.org/10.1101/2023.02.07.527479
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 09, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 93187
Keywords: ASAPCRN, Single-molecule, Single molecules, Microscopy, Imaging, Fluorescence, Fluorescence microscopy, POLCAM, Nile Red, TAB-PAINT, PAINT, Alpha-synuclein fibrils
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000509
Abstract
This is a protocol for the preparation of a sample of alpha-synuclein fibrils on a PLL-coated cover glass for TAB-PAINT imaging with Nile Red. This protocol was used to generate the data shown in Figure 3 of the following publication:

Protocol
Protocol
1d 0h 55m
Prepare alpha-synuclein fibrils by diluting alpha-synuclein monomer Concentration70 micromolar (µM) in PBS (with 0.01% NaN3) and incubating at Temperature37 °C in a shaker (200 rpm) for over Duration24:00:00 . You can store the fibrils at Temperature4 °C for later use.

1d
Argon plasma clean cover glass (VWR collection, 631-0124) for Duration00:30:00 in a plasma cleaner (Expanded Plasma Cleaner, PDC-002, Harrick Plasma).
30m
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 Concentration1 nanomolar (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 Amount70 µL of 0.01% PLL (0.01% poly-L-lysine solution, P4707, Sigma-Aldrich) into the well and wait for Duration00:15:00 . The PLL will coat the surface of the cover glass.
Note
Always use a freshly thawed aliquot of PLL. You can aliquot the PLL and store it in a -80 °C freezer.

15m
Use a pipet to remove the excess PLL from the well and immediately replace it with Amount70 µL of filtered PBS.
Use a pipet to remove the excess filtered PBS from the well and replace withAmount70 µL 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 Amount50 µL of 0.1 µm diameter TetraSpeck Microspheres (0.1 µm, fluorescent blue/green/orange/dark red, T7279, Invitrogen) into the well.
Note
The concentration of TetraSpeck Microspheres (0.1 µm, fluorescent blue/green/orange/dark red, T7279, Invitrogen) will vary between batches from the vendor. You will likely need to test out a few dilutions to find the concentration that results in the right amount of microspheres per field of view.

Gently pipet up and down a few times and wait for Duration00:05:00 .

5m
Use a pipet to remove the excess solution from the well and replace withAmount70 µL 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 Amount50 µL of alpha-synuclein fibrils and pipet up and down a few times in each corner of the sample well and wait for Duration00:05:00
Note
A 1:2 dilution of the fibrils is usually a good starting point, i.e. a 35 mM equivalent monomer concentration.

5m
Remove excess solution using a pipet and add Amount50 µL of filtered PBS to the well.
Remove excess solution again and replace by Amount50 µL of Concentration1 nanomolar (nM) Nile Red.
Image the sample straight away and make sure it doesn't dry out during imaging.