TITLE: Expansion microscopy with R1441C LRRK2 MEF cells: visualization of Myc-RILPL1 and TMEM55B
AUTHORS: Chloe A Hecht, Shahzad S. Khan, Sreeja Nair, Claire Y Chiang, Suzanne R Pfeffer

[DESCRIPTION]
Expansion microscopy is a super-resolution imaging technique that uses expandable hydrogels to increase the physical distance between fluorophores from fixed cells on coverslips. By isotropically expanding samples, the traditional wavelength-limited confocal microscopy imaging techniques can be improved upon ~4X in resolution. Here we use expansion techniques developed previously by others to confocal image TMEM55B and RILPL1. See below for more details and prior methods.

 

 


[STEPS]
SECTION: Transfection of Myc-RILPL1 in LRRK2 R1441C MEF cells
1. Seed LRRK2 R1441C MEF cells at 50-60% confluency on 12 mm glass coverslips in a 24 well plate in 500 µL of complete DMEM (DMEM containing 10% FBS and 1% penicillin-streptomycin) 24 hours before transfection. 

SECTION: Transfection of Myc-RILPL1 in LRRK2 R1441C MEF cells
2. Transfect cells with Myc-RILPL1 plasmid using FuGENE 6 transfection reagent (E2691) at a 3:1 (3 µl FuGENE:1µg plasmid) ratio according to manufacturer’s guidelines. 
SECTION: Transfection of Myc-RILPL1 in LRRK2 R1441C MEF cells
3. Allow cells to attach on coverslips in a 37 °C incubator with 5% CO2 for ~1920 min.

SECTION: Transfection of Myc-RILPL1 in LRRK2 R1441C MEF cells
4. For MLi-2 conditions, add 200 nanomolar (nM) MLi-2 and leave cells in the incubator for 60 min before continuing.

SECTION: Expanding cells in gels
5. Aspirate media and wash cells on coverslips by performing three quick 1 mL washes with PBS.
SECTION: Expanding cells in gels
6. Fix cells by incubating them in PBS containing 4% PFA for 10 min at Room temperature.

SECTION: Expanding cells in gels
7. Incubate cells in AA-FA solution 960 min at 37 °C. 
During this incubation, thaw monomer solution (MS) and keep it on ice. Pre-cool gelation chamber at 4ºC (we use an old 1 mL pipette-tip box with added water for humidity or a 10 cm Petri dish with moistened Kimwipes and Parafilm), and prepare a 10% TEMED and a 10% APS solution diluted in DI water. Keep all solutions on ice .

SECTION: Expanding cells in gels
8. To make Gelation solution (80 µl per coverslip; recipe below makes enough for ~5 coverslips):
    i. Monomer solution                                                   378 µL
    ii. Add 10% TEMED (final 0.5%)                                 21 µL
    iii. ADD 10% APS (final 0.5%)                                     21 µL
 


SECTION: Expanding cells in gels
9. In the cold room, add 80 µL droplets of gelation mixture onto parafilm in a pre-cooled gelation chamber. 
 


SECTION: Expanding cells in gels
10. Place coverslip (cell-side facing down) onto droplet making sure the coverslip is parallel to the parafilm. Wait 5 minutes in the cold and then transfer the chamber containing the coverslips to a 37 °C room for 60 min Be careful not to disturb the coverslip and gel.

SECTION: Expanding cells in gels
11. Gently remove coverslips and gel from the parafilm and place each coverslip in a 6cm dish containing 5 mLdenaturation buffer.
 


SECTION: Expanding cells in gels
12. Incubate gels and coverslips fully submerged in denaturation buffer for 15 min at room temperature with gentle rotation. This step is necessary to cleanly remove gels from the coverslips.

SECTION: Expanding cells in gels
13. Place each gel into a 1.5 mL tube also filled with 1mL fresh denaturation buffer. Incubate at 95 °C using a heat block for 90 min.

SECTION: Expanding cells in gels
14. Remove gels from the 1.5 mL tube and place them each into a 10 cm petri dish with enough DI water to completely cover the gel (~10mL). Incubate the gels twice for 30 min per incubation on a gentle rotator at Room temperature, replacing the water each time. Perform a final incubation with fresh DI water 30 min at Room temperature with gentle rotation.

SECTION: Expanding cells in gels
15. The next day, gels should appear expanded. Incubate gels with enough PBS to completely cover the gel (~10mL) three times for 30 min each time at Room temperature with gentle rotation. Gels should shrink significantly, but will not shrink back to their original size.

SECTION: Expansion Microscopy to Visualize Myc-RILPL1 and TMEM55B
27. 
SECTION: Antibody Staining to Visualize Myc-RILPL1 and TMEM55B
27. 
SECTION: Antibody Staining to Visualize Myc-RILPL1 and TMEM55B
16. Incubate gels with primary antibody (anti-TMEM55b (1:500; 2µl); Anti-Myc (1:250; 4µl) diluted in 1 mL PBS containing 2% BSA) using a 1.5 mL tube 30 min at Room temperature on a rotator. 

SECTION: Antibody Staining to Visualize Myc-RILPL1 and TMEM55B
17. Wash gels three times with enough PBS-T (PBS + 0.1% Tween20) to completely cover the gel (~10mL) for 15 min each time at Room temperature using a 10 cm petri dish with gentle rotation.

SECTION: Antibody Staining to Visualize Myc-RILPL1 and TMEM55B
18. Incubate with secondary antibodies (Donkey anti rabbit 568 (1:500; 2µl), donkey anti-mouse 488 (1:500, 2µl) diluted in 1 mL PBS containing 2% BSA) with gentle rotation 15 min at Room temperature in a 1.5 mL tube.

SECTION: Antibody Staining to Visualize Myc-RILPL1 and TMEM55B
19. Wash gels three times with enough PBS-T (0.1% Tween20) to completely cover the gel (~10 mL) for 15 min each at Room temperature using a 10 cm petri dish with gentle rotation.

SECTION: Antibody Staining to Visualize Myc-RILPL1 and TMEM55B
20. Wash gels twice with enough DI water to completely cover gel (~10mL) for 30 min each at Room temperature, rotating gently during each wash.

SECTION: Antibody Staining to Visualize Myc-RILPL1 and TMEM55B
21. Incubate gels with enough fresh DI water to completely  cover gel (~10mL) 30 min at Room temperature for a final expansion rotating gently. 

SECTION: Antibody Staining to Visualize Myc-RILPL1 and TMEM55B
22. Place gels (cell side down) in an 6-well chamber for confocal imaging.
 

SECTION: Antibody Staining to Visualize Myc-RILPL1 and TMEM55B
23. Image gels!