Oral Intubation of Adult Zebrafish to Administer Experimental Compounds

プロトコル

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board

1. Solutions Required

1. Prepare 150 mg/L (for anesthesia) or 300 mg/L (for euthanasia) of ethyl 3-aminobenzoate methanesulfonate (MS-222) solution with water from the aquarium where the zebrafish are maintained. Fill a small tank with 1 L of anesthetic solution and keep it aerated.
2. Fill another small tank with 1 L of aquarium water without MS-222 for fish recovery and keep it aerated.
3. Make 50 mL of 1x PBS from a 10x sterile stock solution.
4. For cytometry analysis/intestinal cell isolation, prepare enough fresh 0.15% collagenase Type IV solution for 1 mL per fish from a stock solution or from powder in Dulbecco's modified eagle medium (DMEM) with 1% v/v penicillin and streptomycin (see materials). Make aliquots (1 per fish) of 1 mL in 2 mL centrifuge tubes. Keep at 4 °C until 30 min before the dissection step.
5. For confocal microscopy/sample fixation, prepare 50 mL of fresh 4% paraformaldehyde (PFA) solution in PBS or thaw a stock solution from a -20 °C freezer in a fume hood.
   CAUTION: PFA is toxic. Please read the material safety data sheet before working with it. Gloves and safety glasses should be worn, and always leave solutions inside a fume hood.

2. Preparing the Equipment for Oral Intubation

1. Place approximately 1 cm of a fine silicone tube on a 31 G Luer lock needle to cover the needle tip.
2. Cut a 10 µL sterile filter pipette tip (around 2 cm), take the finer end, and place it over the silicone tube as a sheath. Make sure the pipette extends beyond the tip of the needle to avoid injuring the animal.
3. Attach the needle to a 100 µL Luer lock syringe.
   NOTE: Always rinse with ethanol and then phosphate-buffered saline (PBS, see Materials) thoroughly between treatments.

3. Preparing the Fluorescent Nanoparticle Suspension

1. Label the protein nanoparticle with Atto-488 NHS ester (see Table of Materials) or an appropriate fluorescent dye according to the manufacturer's instructions.
2. Resuspend the nanoparticles in 0.1 M sodium bicarbonate buffer at the concentration of 2 mg/mL.
3. Dissolve the Atto 488 NHS ester in amine-free dimethyl sulfoxide (DMSO) at 2 mg/mL. Keep an aliquot of 10 µL to check labeling efficiency.
4. Mix the nanoparticles and the Atto 488 NHS ester at a molar ratio 1:2 (protein: dye) by stirring in the dark.
5. Spin down the labeled nanoparticles by centrifugation at 8,000 x g for 10 min at room temperature, remove the supernatant, and keep it to check labeling efficiency.
6. Wash the labeled nanoparticles by resuspending them in 1 mL of 0.1 M sodium bicarbonate buffer by vortexing and pipetting up and down. Then discard the supernatant by centrifugation at 8,000 x g for 10 min at room temperature. Repeat step 3.6 for 5 times.
7. Resuspend the pellet in 5 mL of 0.1 M sodium bicarbonate buffer in a 15 mL centrifuge tube and make aliquots of the fluorescent nanoparticle in 1.5 mL centrifuge tubes (30 aliquots). Spin down at 8,000 x g for 10 min at room temperature, discard the supernatant, and store at -80 °C protected from the light.

 4. Zebrafish Anesthetization and Oral Intubation

1. Fast the fish (>0.5 g) at least 48 hours before the experiment to empty the intestine.
2. Move the fish (12 fish) to the experimental tanks (6 L) one night before the experiment to allow acclimatization12.
3. Vortex the nanoparticle solution well (e.g., 2,500 rpm and 30 s) and draw up the desired volume of nanoparticle suspension (e.g., 20–50 µL) into the syringe attached to the protected needle.
4. Place the fish in the aerated 150 mg/L MS-222 solution (see section 1) until they sink to the bottom of the tank and do not respond to a tail fin pinch; this process takes less than 5 min.
5. Quickly transfer the anesthetized fish with a net to a wet plastic tray, orientate the animal horizontally to face the needle and immediately start the oral intubation.
6. Carefully support the fish with one hand and open the mouth with the other hand using the protected needle. Gently insert the needle down the esophagus to about 1 cm from the mouth opening.
   NOTE: The operator may feel a slight resistance when the end of the pipette tip has passed the gill. Take care not to angle the needle entry too much which may perforate the gill.
7. Slowly inject the nanoparticle suspension into the fish. Make sure the suspension does not flow outward through the gills or mouth.
8. Gently remove the needle and place the fish into the recovery tank (see section 1). Recovery usually takes 1 minute.
9. Check the fish carefully for any abnormality (e.g., bleeding at the gills is a sign of perforation).
10. Once the fish have recovered, return them to the experimental tanks.

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資料

Name	Company	Catalog Number	Comments
Silicone tube	Dow Corning	508-001	0.30 mm inner diameter and 0.64 mm outer diameter
Luer lock needle	Hamilton	7750-22	31 G, Kel-F Hub
Luer lock syringe	Hamilton	81020/01	100 μL, Kel-F Hub
Filtered pipette tip	Nerbe Plus	07-613-8300	10 μL
MS-222	Sigma Aldrich	E10521	powder
10x PBS	Sigma Aldrich	P5493
Filter paper	Filter-Lab	RM14034252
Atto-488 NHS ester	Sigma-Aldrich	41698
DMEM	Gibco	31966	Dulbecco's modified eagle medium
Maxwell RSC simplyRNA Tissue Kit	Promega	AS1340