Protein Phase Separation Assay: An Optogenetic Method for Mutant RNA-Binding Protein Phase Separation in Spinal Motor Neurons of Zebrafish Larvae

For imaging of the zebrafish larvae expressing optogenetic TDP-43, dechorionate the double-transgenic fish, and anesthetize them in E3 buffer containing 250 micrograms per microliter of Tricane.

Next, pre-heat 1% low-melting temperature agarose containing 250 micrograms per microliter of ethyl 3-aminobenzoate methanesulfonate at 42 degrees Celsius, then, put a drop of the agarose on the glass base dish. The diameter of the dome-shaped agarose drop on the glass dish should be 8 to 10 millimeters.

Next, using a Pasteur pipette, add the anesthetized fish to the agarose on the glass base dish. Minimize the amount of E3 buffer added to the agarose along with the fish. Then, mix by pipetting a few times.

During solidification of the agarose, maintain the fish on its side using a syringe needle, such that the spinal cord is in an appropriate horizontal position. After the agarose has solidified, add a couple drops of E3 buffer onto the dome-shaped agarose-mounted fish.

Using a confocal microscope equipped with a 20x water immersion objective lens, acquire serial confocal z-sections of the spinal cord. Include the cloaca on the ventral side of the fish in the regions of interest as a reference to identify and compare the spinal segments across the time points.

As soon as the imaging is complete, use a syringe needle to carefully crack the agarose and remove the fish from the agarose. Keep the amount of time the fish is embedded in the agarose as short as possible, although agarose embedding for less than 30 minutes does not affect the viability of the fish.

Add 7.5 milliliters of E3 buffer to one well of a 6-well dish, and place the imaged double-transgenic fish into that well. Then, place the dish on the LED panel, keeping the dish, and LED panel 5 millimeters apart with a spacer, and turn on the blue LED light.

Keep some double-transgenic fish in a separate 6-welled dish covered with aluminum foil for unilluminated control fish. After the desired illumination time, image the spinal cord of the illuminated fish as demonstrated earlier.