TITLE: Sample Preparation and 3D-SIM fixed-cell imaging of FTH1 and Filipin in NPC1 and NPC2 mutants
AUTHORS: Felix Kraus

[DESCRIPTION]
Protocol for the evaluation of pUb kinetics using 3D-SIM

[STEPS]
SECTION: Seeding of HeLa cells
1. Wash HeLa TMEM192-3xHA control and NPC1, NPC2 mutants with 1xPBS
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 onto 18x18mm Marienfeld Precision cover glasses thickness No. 1.5H (tol. ± 5 μm).
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 growth medium 
SECTION: Staining
8. Aspirate DMEM and fix cells in warm paraformaldehyde 3% Glutaraldehyde 0.35% in 0.1M Sodium Cacodylate, pH 7.4Aspirate PFA solution and wash wells 3x with PBST (1x PBS, 0.02% Tween 20). 
PFA is toxic and discard in the appropriate waste streams.
SECTION: Staining
10. Permeabilize the cells by adding 0.2% Triton X-100 in PBS.
SECTION: Staining
11. Remove the detergent solution by aspiration.  Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
12. Block cells for 10 min with 3% BSA – 1x PBS.
SECTION: Staining
13. Remove BSA solution by aspiration.  Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
14. Incubate with primary antibodies in 3% BSA - 1x PBS over night at 4°C with gentle shaking.  
a.     anti-FTH1 (rabbit)

SECTION: Staining
15. Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
16. Incubate with secondary antibodies in 3% BSA - 1x PBS for 45 min – 1h.  
a.     Goat anti-rabbit AlexaFluor 568
SECTION: Staining
17. Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
SECTION: Staining
18. Wash coverslips with 1x PBS and mount in Vectashield (Vector Laboratories, H-1000-10) on glass slides.Exchange PBST with 1x PBS and keep cells at 4°C until imaging. Image within the next few days. 
SECTION: Fixed-cell 3D-SIM microscopy
19. Image cells on DeltaVision OMX v4 using an Olympus 60x / 1.42 Plan Apo oil objective (Olympus, Japan). Record 405, 488 and 568 channels using a front-illuminated sCMOS (PCO Photonics, USA) in 512x512px image size mode, 1x binning, 125 nm z-stepping and with 15 raw images taken per z-plane (5 phase-shifts, 3 angles).
SECTION: Fixed-cell 3D-SIM microscopy
20. Reconstruct raw images using CUDA-accelerated 3D-SIM reconstruction code (https://github.com/scopetools/cudasirecon) based on Gustafsson et al. (2008[FK1] ). The Optimal optical transfer function (OTF) was determined via an in-house build software, developed by Talley Lambert from the NIC / CBMF (GitHub: https://github.com/tlambert03/otfsearch, all channels were registered to the 528nm output channel, Wiener filter: 0.002, background: 90).   
SECTION: 3D-redendering in ChimeraX
21. Open .dv composite image stacks in Fiji. Separate channels and save them as separate .tiff stacks for import into ChimeraX
SECTION: 3D-redendering in ChimeraX
22. Open ChimeraX and open one of the two image stacks into a new project. 
SECTION: 3D-redendering in ChimeraX
23. While not moving the scene, drag the second tiff stack into ChimeraX. The two channels should thus keep their previous alignment. 
SECTION: 3D-redendering in ChimeraX
24. Go Tools > Volume Data > Volume Viewer. 
Change the representation of both channels to surface or mesh (or what fits your visualization needs best). 
Adjust color and levels of the image on the histogram. 
SECTION: 3D-redendering in ChimeraX
25. In the Volume Viewer tab, use the "Hide Dust" function to remove unspecific background specs / spots from the render. Repeat for both channels. 
SECTION: 3D-redendering in ChimeraX
26. Move the render into an adequate position and export the image. 
SECTION: 3D-redendering in ChimeraX
27. Import the resulting image into illustrator for making your figure.
SECTION: Staining
9. Stain cholesterol with Filipin (0.05mg/ml in PBS / 10% FBS) for 2h at RT. 
Wash cells after staining with PBST for 2 min.