Single-molecule pulldown for immunodetection of protein aggregates

Treat coverslips with argon plasma for 45 mins

Immerse coverslips in 0.22 micron-filtered 1M potassium hydroxide for 20mins

Rinse coverslips in ultrapure deionised water and immerse in APTMS solution (prepared as 3.75 mL glacial acetic acid, 1 mL APTMS, 75 mL methanol) and incubate covered for 20 mins at RT.

Rinse coverslips sequentially in methanol and ultrapure deionised water and use fast flowing argon to dry slides rapidly. Inspect slide to ensure visually clean.

Attach 18-well Ibidi sticky-slide chamber to coverslip, apply pressure to ensure fully adhered.

Dispense 50 uL mPEG solution per well (95mg/mL mPEG-SVA, 5 mg/mL biotin-PEG, 0.1M sodium hydrogen carbonate), parafilm seal chamber and incubate covered overnight at RT.

Empty PEG solution by inversion and wash wells with ultrapure deionised water x3

Dry wells thoroughly using fast-flowing argon gas

Apply 50 uL 0.2 mg/mL streptavidin prepared in 0.02-micron filtered T50 buffer (10 mM Tris pH 8.0 supplemented with 50 mM sodium chloride) per well and incubate covered for 10mins at RT

Wash wells with 0.02-micron filtered T50 buffer x3

Apply 50 uL of 100 nM biotinylated capture antibody and incubate covered for 20 mins at RT

Wash wells with 0.02-micron filtered PBS x3

Apply 50 uL neat CSF per well and incubate covered for 24 hours at RT

Wash wells with 0.02-micron filtered PBS x3

NB. CSF is biohazardous, aspirate to remove liquid waste instead of slide inversion

Apply 50 uL 2nM fluorophore-labelled detection antibody and incubate covered for 3 hours at RT

Wash wells with 0.02-micron filtered PBS x3

Apply 50 uL PBS per well and image immediately

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 10 uM working stock in 0.02-micron filtered PBS

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 sequentially excited at 638nm and 488 nm, 20 frames captured per field in each channel at 20 frames s^-1.

On completion of imaging one well, return to position 0 (based on metadata values), cautiously apply 50 uL 10 uM ThT and re-image all fields with 405nm excitation.