This method can help answer key questions in the mucosa vaccinology field, such as how intestinal mucosa uptake biological compounds and how such biologicals modulate the immune system at local site after intubation. The main advantage of this technique is that it's simple to set up, quick, and shows low mortality rates. This method can provide insight into mucosal vaccinology.
It can also be applied to other compounds, such as chemicals, toxics, or pathogens. Visual demonstration of this method is critical as in the oral intubation and intestinal dissection steps, you need to be careful because in intubation you could injure the zebrafish and cause death. In dissection, the end of the intestine is very fine and fragile.
Place approximately one 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 one 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 has recovered, return it to one of the experimental tanks.
After euthanizing the fish according to the text protocol, place the fish on a piece of filter paper. Using sharp dissection scissors, make a semi-circular incision from the anus to the operculum, and open the incision with fine tweezers. Then, cut both ends of the intestine, take out all of the internal organs, and place them on the filter paper.
Next, separate the intestine from the internal organs, and stretch it out, taking care to obtain all of the intestine. Finally, use tweezers to roll the intestine on the filter paper to detach the adhesive tissue. Using this protocol, zebrafish of mixed sex were successfully intubated with different recombinant protein nanoparticles.
Using the homemade oral intubation device, a low average percentage of mortality was achieved. To evaluate the delivery of the fluorescent nanoparticles inclusion bodies made with recombinant TNF alpha, a cytometry analysis was performed. The total amount of fluorescent cells was significantly higher in the nanoparticle-intubated group than in the control group both five and 24 hours after intubation.
The intubation equipment is inexpensive, precise, and reusable. The whole intubation procedure can be done very quickly by one operator, and, importantly, the method is consistent between different operators. The combination of our intubation method with downstream analysis could be ideal for oral vaccine development.
