Detailed zebrafish Smurf gavage protocol

Cut the gavage tube to approximately 2.5 centimeters in length. After the fish is anesthetized, confirm the tube size is appropriate and adjust the length as needed to match the individual fish's size.

Aliquot 5 microliters of saline HBSS onto parafilm and aspirate it into a 30 gauge gavage syringe, avoiding excessive air. Carefully insert the syringe into the tubing without tearing it.

Allow the fish to breathe anesthetic solution (4% tricane) until it is fully sedated. Place the fish onto a pre-soaked sponge that has been cut to accommodate the fish's size, protecting the gills.

Measure the gavage tube against the fish to determine the appropriate insertion depth to reach the intestinal bulge. This measurement guides how far the tube should be inserted during the procedure.

Gently open the fish's mouth and insert the tube carefully, feeling for any resistance. Slowly advance the tube to the previously measured depth, adjusting if needed until it reaches the target location.

While holding the tube steady with one hand, use the other hand to press the plunger firmly to deliver the saline solution. Continue applying pressure while gradually withdrawing the tube to ensure complete delivery.

After tube withdrawal, inspect the mouth and gills for any signs of bleeding or injury. Transfer the fish to a recovery container with fresh water and allow it to recover from anesthesia.