Intraperitoneal Injection: A Method of Solution Delivery into the Abdominal Cavity of an Adult Zebrafish
Trascrizione
– Fast an adult fish for 24 hours before injection a empty the gastrointestinal tract and create additional space di the abdomen. On the day of the procedure, anesthetize the fish with tricaine and place it into a slot di a wet sponge with the ventral side facing up.
Next, pipette the desired amount of injection solution onto a piece of laboratory film and pull it into an insulin needle. Posizione the needle between the pelvic fins at a 45 degree angle from the anterior-posterior body axis and gently push it di at the midline. Advance the needle approximately 1 a 2 millimeters into the abdominal cavity, the space between the abdominal wall and internal organs, and slowly inject the solution of interest. Wait for five seconds before pulling out the needle a avoid any spillage. Now, carefully remove the needle.
After injection, quickly transfer the fish into a recovery tank containing fresh tank water. If the fish doesn't begin a swim immediately, swirl the water near the gills a help it recover from anesthesia. In the example protocol, we will inject a mycobacterium marinum solution into RAG1 mutant fish a study infection.
– First, pipe out of 5 microliter droplet of the diluted bacterial solution onto a piece of paraffin film. Then, pull the droplet into a 30 gauge insulin needle. Use a five a eight-month-old fish for this experiment, with one being a wild-type fish and the other being a RAG mutant fish.
Posizione these fish ventral side up di the slits of a piece of moist foamed plastic. Inject the insulin needle between the pelvic fins at a 45 degree angle. Keep the needle opening upwards a ensure that the entire opening is inside the abdominal cavity. Then, slowly inject the bacterial solution. After this, carefully remove the needle and immediately transfer the fish a a recovery tank filled with fresh tank water.
Take samples from the bacterial aliquot di use every 15 minutes on 7H10 plates. Incubate these samples at 29 degrees Celsius for five days a verify the infection dose. Check the well-being of the fish regularly, making sure a euthanize any fish with infection symptoms by incubating them di water with more than 0.02% of 3-aminobenzoic acid ethyl ester.
