SAVE imaging of protein aggregates in cerebrospinal fluid

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.

Steps

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

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

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.

Preparation of ThT solution

12.

Add 70 uL of PLL to each chamber and incubate for 30 minutes.