Nov 15, 2023
SAVE imaging of protein aggregates in cerebrospinal fluid
- 1EaStCHEM School of Chemistry, University of Edinburgh, Edinburgh, UK.;
- 2IRR Chemistry Hub, Institute for Regeneration and Repair, University of Edinburgh, Edinburgh, UK.
Protocol Citation: mathew.horrocks 2023. SAVE imaging of protein aggregates in cerebrospinal fluid. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldmqkol5b/v1
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: November 15, 2023
Last Modified: November 15, 2023
Protocol Integer ID: 90959
Abstract
This protocol describe the procedures used to clean a coverglass surface and perform ThT detection of protein aggregates in CSF (SAVE imaging) on a TIRF microscope.
Materials
Equipment:
- Diener Electronic ZeptoOne Plasma Cleaner
- Oxford Nanoimager
Consumables:
- Coverslips (Merck, C9056-1CS)
- 0.02-micron syringe filters (Merck, WHA68092002)
- Frame-Seal slide chambers (9 × 9 mm2, Biorad, Hercules, CA, SLF-0601).
- Eppendorf Protein LoBind tubes (Merck, EP0030108094)
Reagents:
- Phosphate buffered saline (Merck, P4417)
- Thioflavin T (Merck, 596200-500MG)
- Ethanol (Sigma-Aldrich, 459836)
- Poly-l-lysine (70 000–150 000 molecular weight, Sigma-Aldrich, P4707-50 ML)
Preparation of ThT
Preparation of ThT
Prepare approx. 4 mM stock of ThT in 100% ethanol and vortex extensively (approx. 1 hour).
Prepare approx. 200 uM ThT dilution in PBS, vortex thoroughly (approx. 20 mins) and filter through 0.02-micron filter.
Measure concentration of ThT preparation using a DeNovix spectrophotometer (extinction coefficient 36,000 M-1 cm-1 at 412 nm).
Prepare a 50 uM working stock in 0.02-micron filtered PBS
Addition of sample for imaging
Addition of sample for imaging
Add 5 uL CSF, 5 uL ThT working stock, and 40 uL PBS to an eppendorf tube.
Add 50 uL of the sample prepared in the previous step to the PLL-treated coverslips, and incubate for 1h.
Rinse with PBS.
Add 100 uL of 5 uM ThT to the coverslip.
Imaging
Imaging
Acquire an 8 x 8 grid of 200-micron spaced fields of view per well by total internal reflection fluorescence microscopy using the ONI Nanoimager with 100x/1.4 oil immersion objective lens. Samples are excited at 405 nm, 50 frames captured per field in each channel at 20 frames s-1.