TITLE: Fixation and imaging of HeLa cells after mitochondrial depolarization
AUTHORS: OLIVIA HARDING, Erika L.F. Holzbaur

[DESCRIPTION]
Ectopic expression of p62/SQSTM1 often induces puncta formation and poor cell health due to p62’s proclivity to multimerize and form filaments. We used the cell fixation protocol described here in order to over-express fluorescently tagged p62 at low levels in p62-/- cells and amplify the signal with immuno-labeling. By this method, we were able to express a variety of tagged p62 mutations to determine their effects on NEMO recruitment to depolarized mitochondria without introducing toxic artifacts of p62 overexpression. We also note the use of a modified fixation protocol to visualize various endogenous ATG8 proteins, which are also difficult to over-express in our system. Thus, fixation techniques are a critical complement to studies in live cells. 

[BEFORE_START]

The start point for this protocol is after cells grown on glass coverslips in a 12-well plate have been transfected with Parkin, EGFP-NEMO, HaloOPTN, and mCherry-p62 for 18- 1440 min and tagged with Halo ligand.
Prepare 45 millimolar (mM) stock of Antimycin A by suspending 50 mg solid AntA in 2 mL ethanol.
 Prepare 10 millimolar (mM) stock Oligomycin A by suspending 5 mg solid OligA in 630 µL DMSO
Prepare 4% PFA in PBS. Keep frozen at -20 °C.
 
 
Prepare Culture Media by making 10% FBS, 1% GlutaMAX solution in DMEM.
Prepare 0.5% Triton X-100 in PBS (Permeabilization buffer).
 

Prepare 0.2% Triton X-100/3% BSA in PBS (Blocking buffer).
 

  Prepare a humidity chamber as previously described (dx.doi.org/10.17504/protocols.io.bujsnune)









[GUIDELINES]

This protocol was adapted from a previous protocol for similar techniques  
      (see dx.doi.org/10.17504/protocols.io.bujsnune)
Here we use a primary antibody to a mitochondrial protein, HSP60, in order to identify mitochondria, instead of employing a genetically encoded fluorescent protein. While there are excellent mitochondrially-targeted fluorescent proteins, using anti-HSP60 allows us to demonstrate the versatility of the protocol to image both expressed, tagged proteins and immune-labeled structures.

[STEPS]
SECTION: AntA/OligA treatment
1.  
Prepare working AntA/OligA solution by transferring 0.5 mL conditioned media to a 1.5 mL tube and adding 0.25 µL 10 millimolar (mM) OligA and 2 mL 45 millimolar (mM) AntA.
 
SECTION: AntA/OligA treatment
2. Gently drop working AntA/OligA solution onto cells.
SECTION: AntA/OligA treatment
3.  Incubate at 37 °C, 5% CO2 for 55 min- 65 min.
 

SECTION: Fixation
4. 
25 minutes before AntA/OligA treatment is complete, warm 4% PFA and 1X PBS to 37 °C.
 
SECTION: Fixation
5. Remove cells from incubator and aspirate media.
 

SECTION: Fixation
6. Quickly drop on 0.5 mL warmed 1X PBS.

SECTION: Fixation
7. Aspirate PBS.
SECTION: Fixation
8.  Repeat warm PBS wash.
SECTION: Fixation
9. Drop on 1 mL warmed 4% PFA.

SECTION: Fixation
10.  Incubate at 37 °C for 10 min.
 



SECTION: Fixation
11. Drop on 0.5 mL warmed PBS.

SECTION: Fixation
14. Incubate covered at Room temperature for 3 min.
 



SECTION: Permeabilization
15.  
 Aspirate PBS.
 
SECTION: Permeabilization
16. Drop on 0.75 mL Permeabilization buffer.

SECTION: Permeabilization
17.  Incubate covered at Room temperature for 5 min.
 



SECTION: Blocking
18.  
 Add ~250 µL Blocking buffer to each well.
 
SECTION: Blocking
19. Incubate covered at Room temperature for 45 min.
 

SECTION: Blocking
20. Aspirate Blocking buffer.
SECTION: Primary antibody
21.  
Prepare 200 µL Primary antibody dilution per coverslip, with 1:250 antiHSP60 and 1:500 anti-p62 in Blocking buffer.
 

SECTION: Primary antibody
22. Use sharp forceps to carefully lift the coverslip out of the well and dab excess Blocking buffer on a Kimwipe.
SECTION: Primary antibody
23. Place slip cell-side up on Parafilm in the humidity chamber.
SECTION: Primary antibody
24.  From the edge of the coverslip, pipet primary antibody dilution onto the cells.
SECTION: Primary antibody
25. Incubate covered at 4 °C 5 min.
 



SECTION: Primary antibody
26. 18- 24 hours later, aspirate antibody dilution from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.

SECTION: Primary antibody
27. From the edge of the coverslip, pipet 100 µL Room temperature PBS onto the cells.

SECTION: Primary antibody
28.  Incubate covered at Room temperature for 5 min.

SECTION: Primary antibody
29. Aspirate PBS from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.
SECTION: Primary antibody
30. Repeat previous three steps for a total of four washes.

SECTION: Secondary antibody
31.  
 Prepare secondary antibody solution by diluting goat anti-rabbit 405 and goat anti-mouse 1:200 in Blocking buffer.
 

SECTION: Secondary antibody
32. From the edge of the coverslip, pipet secondary antibody dilution onto the cells.
SECTION: Secondary antibody
33.  Incubate covered at Room temperature for 45 min.
 

SECTION: Secondary antibody
34. Aspirate antibody dilution from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.
35. From the edge of the coverslip, pipet 100 µL  Room temperature PBS onto the cells.
 
36. Incubate covered at Room temperature for 5 min.

37. Aspirate PBS from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.
38. Repeat previous three steps for a total of four washes.
SECTION: Mounting
39.  
Pipet 12.5 µL Room temperature VectaShield onto a microscope slide.
 
SECTION: Mounting
40. Pick up coverslip with forceps and dab excess PBS onto a Kimwipe.
SECTION: Mounting
41. Lay coverslip cell-side down onto VectaShield drop.
SECTION: Mounting
42. Seal edges of coverslip with nail polish.
SECTION: Mounting
43. Lay flat until set.
 

SECTION: Imaging
44. Using 60X objective and RFP epifluorescence, find the focal plane of fixed cells by looking for dsRed2-labeled mitochondria.
 

SECTION: Imaging
45. Configure 405, 488, 561, and 647 lasers and accompanying exposure times to maximize dynamic range of each signal.
 

SECTION: Imaging
46. Configure collection parameters to record a >2 um Z stack of images.
 

SECTION: Imaging
47. Collect images from fields of view with multiple cells exhibiting all visible tags. Collect volume from the 50th to 75th percentile of the cell (see Figure 1). If mitochondria are depolarized, can use OPTN signal (recruitment to
fragmented mitochondria) to determine whether Parkin is expressed.


  

SECTION: Imaging
48. Collect images in order to record 10-20 cells per condition, depending on the quality of the transfection and cells. Collect images from diverse areas of the coverslip.
SECTION: Fixation
12. Aspirate PBS.
SECTION: Fixation
13. Drop on 0.5 mL warmed PBS.

SECTION: Primary antibody
30.1. Wash (1/4):
From the edge of the coverslip, pipet 100 µL Room temperature PBS onto the cells.
Incubate covered at Room temperature for 5 min.
Aspirate PBS from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.
SECTION: Primary antibody
30.4. Wash (4/4):
From the edge of the coverslip, pipet 100 µL Room temperature PBS onto the cells.
Incubate covered at Room temperature for 5 min.
Aspirate PBS from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.
SECTION: Primary antibody
30.3. Wash (3/4):
From the edge of the coverslip, pipet 100 µL Room temperature PBS onto the cells.
Incubate covered at Room temperature for 5 min.
Aspirate PBS from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.
SECTION: Primary antibody
30.2. Wash (2/4):
From the edge of the coverslip, pipet 100 µL Room temperature PBS onto the cells.
Incubate covered at Room temperature for 5 min.
Aspirate PBS from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.
38.1. Wash (1/4)
From the edge of the coverslip, pipet 100 µL  Room temperature PBS onto the cells.
Incubate covered at Room temperature for 5 min.
Aspirate PBS from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.
38.2. Wash (2/4)
From the edge of the coverslip, pipet 100 µL  Room temperature PBS onto the cells.
Incubate covered at Room temperature for 5 min.
Aspirate PBS from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.
38.3. Wash (3/4)
From the edge of the coverslip, pipet 100 µL  Room temperature PBS onto the cells.
Incubate covered at Room temperature for 5 min.
Aspirate PBS from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.
38.4. Wash (4/4)
From the edge of the coverslip, pipet 100 µL  Room temperature PBS onto the cells.
Incubate covered at Room temperature for 5 min.
Aspirate PBS from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.