This article describes a method for delivering nanostructured cytokine inclusion bodies into zebrafish via oral intubation. The process facilitates the study of immune responses triggered by these nanoparticles in a live model organism.
Begin by loading a sterile oral intubation device with a suspension of nanostructured cytokine inclusion bodies — the experimental nanoparticle.
Place a fasting anesthetized zebrafish onto a moist plastic tray.
Hold the fish steadily and gently open its mouth using the pipette tip attached to the oral intubation device.
Carefully insert the needle through the fish's mouth, down the esophagus.
Once the pipette tip passes the gills and encounters a slight resistance, avoid further insertion.
Gently inject the nanoparticles into the fish.
Upon injection, these cytokine nanoparticles come in direct contact with the epithelial cells that line the digestive tract of the zebrafish.
These cells internalize the nanoparticles through endocytosis.
Within the epithelial cell, the vesicle undergoes intracellular processing to release the cytokine nanoparticles.
These cytokine nanoparticles exit into the underlying connective tissue, interact with resident immune cells, and trigger intracellular signaling events, resulting in a cytokine-driven immune response.
Place approximately 1 centimeter of fine silicone tubing on a 31-gauge Luer-lock needle. Cut a sterile 10-microliter filter pipette tip, take the finer end, and place it over the silicone tube as a sheath.
To begin, attach the needle apparatus to a 100-microliter Luer-lock syringe. First, move the fish to the experimental tanks one night prior to the experiment. Vortex the prepared nanoparticle solution well, and draw up the desired volume of the suspension. After safely anesthetizing the fish, use a net to quickly transfer the fish to a wet plastic tray.
Orient the animal horizontally to face the needle. Carefully support the fish with one hand, and open the mouth with the protected needle, then, gently insert the needle down the esophagus to about 1 centimeter from the mouth opening.
It's difficult to see how far the needle should go, and this is a matter of practice. You will feel the needle as it finds a stop, and then, by manipulating the needle, you'll find the correct angle to insert the device a little more.
Slowly inject the nanoparticle suspension into the fish, ensuring the suspension does not flow outward through the gills or the mouth. After this, gently remove the needle, and place the fish into a recovery tank. While the fish recovers, monitor the fish for any abnormalities. Once the fish is recovered, return it to one of the experimental tanks.
繁體中文
