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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board
1. Preparation of LED for blue light illumination
- Turn on an LED panel by using the associated application installed on a tablet/phone. Put the probe of a spectrometer into an empty well of a 6-well dish and adjust the LED light to the wavelength peaking at ~456 nm through the application. Place the optical sensor of an optical power meter in the empty well and adjust the power of the LED light (~0.61 mW/cm2). The LED light setting can be saved and is retrievable in the application.
- Introduce the dish/LED panel setting to the incubator at 28 °C. Finish this step before imaging of fish starts at 48 hpf.
2. Imaging of zebrafish larvae expressing optogenetic TDP-43
- Select Tg[mnr2b-hs:opTDP-43h] Tg[mnr2b-hs:EGFP-TDP-43z] double-transgenic fish at least before 47 hpf, based on RFP (opTDP-43h) or EGFP (EGFP-TDP-43z) fluorescence in the spinal motor column using the epifluorescence microscope equipped with a Plan-Neofluar 5x/0.15 objective lens.
- Dechorionate the Tg[mnr2b-hs:opTDP-43h] Tg[mnr2b-hs:EGFP-TDP-43z] double-transgenic fish.
- Preheat 1% low melting temperature agarose containing 250 µg/mL of ethyl 3-aminobenzoate methanesulfonate salt at 42 °C.
- Briefly anesthetize Tg[mnr2b-hs:opTDP-43h] Tg[mnr2b-hs:EGFP-TDP-43] double-transgenic fish at 48 hpf in E3 buffer containing the same concentration of Tricane.
- Put a drop of the preheated 1% low melting temperature agarose on the glass base dish at the room temperature. The diameter of the dome-shaped agarose drop on the glass dish is 8-10 mm.
- Using a Pasteur pipette, add the anesthetized fish to the low melting temperature agarose on the glass base dish, and then mix by pipetting a few times. Minimize the amount of the E3 buffer added to the agarose along with the fish.
- Maintain the fish on its side by using a syringe needle during the solidification of agarose (typically ~1 min) to ensure that the spinal cord is in an appropriate horizontal position. After the solidification, put a couple of drops of E3 buffer onto the dome-shaped agarose-mounted fish.
- Acquire serial confocal z-sections of the spinal cord by scanning with a confocal microscope equipped with a 20x water immersion objective lens with the numerical aperture 1.00, using a scan speed of 4.0 µs per pixel (12 bits per pixel), a step size of 1.0 µm per slice for the objective, and a combination of excitation/emission wavelengths: Channel 1) 473/510 nm for EGFP and Channel 2) 559/583 nm for mRFP1.
NOTE: The cloaca on the ventral side of the fish is included in the regions of interest (ROI) as a reference, which helps to identify and compare the spinal segments (levels 16-17) across the time points.
- Remove the fish from the agarose by carefully cracking the agarose with a syringe needle as soon as the imaging is complete. Keep the amount of time the fish is embedded in the agarose as short as possible, although the agarose embedding for <30 min does not affect the viability of the fish.
3. Light stimulation of opTDP-43h-expressing fish by field illumination of a blue light-emitting diode (LED) light
- Add 7.5 mL of E3 buffer to the well and place the imaged Tg[mnr2b-hs:opTDP-43h] Tg[mnr2b-hs:EGFP-TDP-43z] double-transgenic fish into the well. Place the six-well dish on the LED panel by keeping the dish and LED panel 5 mm apart with a spacer (for example, with five slide glasses stacked).
- Turn on the blue LED light. Keep some of the Tg[mnr2b-hs:opTDP-43h] Tg[mnr2b-hs:EGFP-TDP-43z] double-transgenic fish in a separate six-well dish covered with aluminum foil when unilluminated control fish are necessary (i.e., in dark conditions).
- After the illumination (e.g., for 24 h at 72 hpf in Figure 1), image the spinal cord of the illuminated fish by repeating the steps 2.3 - 2.9.
