Expansion microscopy with R1441C LRRK2 MEF cells: visualization of Myc-RILPL1 and TMEM55B

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

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.

Allow cells to attach on coverslips in a 37°C incubator with 5% CO₂ for ~32h 0m 0s.

For MLi-2 conditions, add 200nanomolar (nM) MLi-2 and leave cells in the incubator for 1h 0m 0s before continuing.

Aspirate media and wash cells on coverslips by performing three quick 1 mL washes with PBS.

Fix cells by incubating them in PBS containing 4% PFA for 0h 10m 0s at Room temperature.

Incubate cells in AA-FA solution 16h 0m 0s 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 .

To make Gelation solution (80 µl per coverslip; recipe below makes enough for ~5 coverslips):

i. Monomer solution                                                   `378µL`



ii. Add 10% TEMED (final 0.5%)                                 `21µL`



iii. ADD 10% APS (final 0.5%)                                     `21µL`

In the cold room, add 80µL droplets of gelation mixture onto parafilm in a pre-cooled gelation chamber.

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 1h 0m 0s Be careful not to disturb the coverslip and gel.

Gently remove coverslips and gel from the parafilm and place each coverslip in a 6cm dish containing 5mLdenaturation buffer.

Incubate gels and coverslips fully submerged in denaturation buffer for 0h 15m 0s at room temperature with gentle rotation. This step is necessary to cleanly remove gels from the coverslips.

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 1h 30m 0s.

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 0h 30m 0s per incubation on a gentle rotator at Room temperature, replacing the water each time. Perform a final incubation with fresh DI water 0h 30m 0s at Room temperature with gentle rotation.

The next day, gels should appear expanded. Incubate gels with enough PBS to completely cover the gel (~10mL) three times for 0h 30m 0s each time at Room temperature with gentle rotation. Gels should shrink significantly, but will not shrink back to their original size.

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 0h 30m 0s at Room temperature on a rotator.

Wash gels three times with enough PBS-T (PBS + 0.1% Tween20) to completely cover the gel (~10mL) for 0h 15m 0s each time at Room temperature using a 10 cm petri dish with gentle rotation.

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 0h 15m 0s at Room temperature in a 1.5 mL tube.

Wash gels three times with enough PBS-T (0.1% Tween20) to completely cover the gel (~10 mL) for 0h 15m 0s each at Room temperature using a 10 cm petri dish with gentle rotation.

Wash gels twice with enough DI water to completely cover gel (~10mL) for 0h 30m 0s each at Room temperature, rotating gently during each wash.

Incubate gels with enough fresh DI water to completely cover gel (~10mL) 0h 30m 0s at Room temperature for a final expansion rotating gently.

Place gels (cell side down) in an 6-well chamber for confocal imaging.

Image gels!