TITLE: Microscopy-based measurements of p62 recruitment in HeLa
AUTHORS: Felix Kraus

[DESCRIPTION]
Protocol for microscopy-based measurements of p62 recruitment in HeLa

[STEPS]
SECTION: Seeding of HeLa cells
1. Wash HeLa cells expressing doxycycline-inducible Parkin with 1x PBS
SECTION: Seeding of HeLa cells
2. Add Trypsin to cells for 5 min and incubate at 37°C to dissociate cells from plastic well
SECTION: Seeding of HeLa cells
3. Resuspend cells in 1 mL DMEM media
SECTION: Seeding of HeLa cells
4. Count cells
SECTION: Seeding of HeLa cells
5. Seed appropriate number of cells into 24-well glass bottom dish
SECTION: Seeding of HeLa cells
6. Top up glass bottom dish with either 1 mL DMEM and place cells back into incubator
SECTION: Seeding of HeLa cells
7. The next day exchange DMEM with DMEM + 2µg/ml doxycycline for 18h to induce Parkin expression.
SECTION: Seeding of HeLa cells
8. Induce mitophagy using Antimycin A / Oligomycin A for the desired time.    Staining
SECTION: Staining
9. Aspirate DMEM and fix cells in 1 ml pre-warmed 4% PFA for 30 min.
SECTION: Staining
10. Aspirate PFA solution and wash wells 3x with PBST (1x PBS, 0.02% Tween 20)
SECTION: Staining
11. Permeabilize the cells by adding 0.2% Triton X-100 in PBS.
SECTION: Staining
12. Remove the detergent solution by aspiration.  Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
13. Block cells for 10 min with 3% BSA – 1x PBS.
SECTION: Staining
14. Remove BSA solution by aspiration.  Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
15. Incubate with primary antibodies in 3% BSA - 1x PBS for 3h at RT with gentle shaking. 
a.     Anti-p62 (mouse)  
b.     Anti-FIP200 (rabbit)
SECTION: Staining
16. Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
17. Incubate with secondary antibodies in 3% BSA - 1x PBS for 45 min – 1h.  
a.     Goat anti-mouse AlexaFlour 488  
b.     Goat anti-rabbit AlexaFluor 568
SECTION: Staining
18. Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
19. Add Hoechst33342 or DAPI 1:2000 to wells for 5 min with gentle shaking.
SECTION: Staining
20. Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
21. Exchange PBST with 1x PBS and keep cells at 4°C until imaging. Image within the next few days.    Fixed-cell microscopy
SECTION: Staining
22. Mount glass bottom plate on Yokogawa CSU-W1 spinning disk confocal on a Nikon Eclipse Ti-E motorized microscope equipped with a Nikon Apochromat 60×/1.42 N.A oil-objective lens. Image signals of 488/568/647 fluorophores in sequential manner with a Nikon LUN-F XL solid state laser combiner ([laser line – laser power]: 488 - 80mW, 561 - 65mW, 640nm - 60mW]) using a Semrock Di01-T405/488/568/647 dichroic mirror. Fluorescence emissions were collected with 488 Chroma ET525/50m [488 nm], 568 Chroma ET605/52m [561 nm], 633 Chroma ET705/72m [640 nm] filters, respectively (Chroma Technologies) using NIS-Elements image acquisition software. Consistent laser intensity and exposure times must be maintained for all samples. Acquire 8 µm z-stacks for each image.
SECTION: Staining
23. Image adequate number of cells per repeat in each condition.    Evaluation
SECTION: Evaluation
24. Perform image quantification was in your tool of choice. Here we will use ImageJ/FiJi and custom-written batch-macros (https://github.com/harperlaboratory/FBXO7).
SECTION: Evaluation
25. Filter p62 signal (Gaussian Blur, sigma=2) and converted images into binary files using the “Intermodes thresholding” method.
SECTION: Evaluation
26. Measure binary file these masks were using the "Analyze Particles..." command (pixel size exclusion: 0.1-30, exclude edge objects).
SECTION: Evaluation
27. Save results image stacks as .csv files, together with the original overlay.tiff file for QC purposes.
SECTION: Evaluation
28. Count number of nuclei for normalization.
SECTION: Evaluation
29. Plot results in your tool of choice for graphing and statistical analysis.