TITLE: Single-molecule pulldown for immunodetection of protein aggregates
AUTHORS: Rebecca Saleeb, Craig Leighton, Ji-Eun Lee, Mathew Horrocks

[DESCRIPTION]
This protocol describe the procedures used to clean and passivate a coverglass surface, functionalise it with biotin and specifically immobilise proteins of interest for detection with single channel imaging (SiMPull), two-colour coincidence aggregate detection (STAPull) or ThT detection (SAVE imaging) on a TIRF microscope. This protocol supports published work (Saleeb et al., 2023). 

[STEPS]
SECTION: Coverslip passivation and functionalisation
1. Treat coverslips with argon plasma for 45 mins
SECTION: Coverslip passivation and functionalisation
2. Immerse coverslips in 0.22 micron-filtered 1M potassium hydroxide for 20mins
SECTION: Coverslip passivation and functionalisation
3. 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.
SECTION: Coverslip passivation and functionalisation
4. Rinse coverslips sequentially in methanol and ultrapure deionised water and use fast flowing argon to dry slides rapidly. Inspect slide to ensure visually clean. 
SECTION: Coverslip passivation and functionalisation
5. Attach 18-well Ibidi sticky-slide chamber to coverslip, apply pressure to ensure fully adhered.
SECTION: Coverslip passivation and functionalisation
6. 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.
SECTION: Sample pull-down and labelling
7. Empty PEG solution by inversion and wash wells with ultrapure deionised water x3
SECTION: Sample pull-down and labelling
8. Dry wells thoroughly using fast-flowing argon gas
SECTION: Sample pull-down and labelling
9. 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
SECTION: Sample pull-down and labelling
10. Wash wells with 0.02-micron filtered T50 buffer x3
SECTION: Sample pull-down and labelling
11. Apply 50 uL of 100 nM biotinylated capture antibody and incubate covered for 20 mins at RT
SECTION: Sample pull-down and labelling
12. Wash wells with 0.02-micron filtered PBS x3
SECTION: Sample pull-down and labelling
13. Apply 50 uL neat CSF per well and incubate covered for 24 hours at RT
SECTION: Sample pull-down and labelling
14. Wash wells with 0.02-micron filtered PBS x3

NB. CSF is biohazardous, aspirate to remove liquid waste instead of slide inversion
SECTION: Sample pull-down and labelling
15. Apply 50 uL 2nM fluorophore-labelled detection antibody and incubate covered for 3 hours at RT
SECTION: Sample pull-down and labelling
16. Wash wells with 0.02-micron filtered PBS x3
SECTION: Sample pull-down and labelling
17. Apply 50 uL PBS per well and image immediately
SECTION: Thioflavin T preparation
18. Prepare approx. 4 mM stock of ThT in 100% ethanol and vortex extensively (approx. 1 hour)
SECTION: Thioflavin T preparation
19. Prepare approx. 200 uM ThT dilution in PBS, vortex thoroughly (approx. 20 mins) and filter through 0.02-micron filter.
SECTION: Thioflavin T preparation
20. Measure concentration of ThT preparation using a DeNovix spectrophotometer (extinction coefficient 36,000 M-1 cm-1 at 412 nm)
SECTION: Thioflavin T preparation
21. Prepare a 10 uM working stock in 0.02-micron filtered PBS
SECTION: Imaging
22. 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. 
SECTION: Imaging
23. 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.