The overall goal of this procedure is to intravenously deliver a test article to a neonate mouse. This is accomplished by first identifying the one to two day old mice to be treated. The second step is to prepare the injection materials.
Next, the injection is performed. The final step is to return the treated and awake mice to their mother. Ultimately, a downstream assay is required to determine whether the test article have the desired effect.
Generally, individuals new to this method struggle most with identifying and targeting the appropriate vein. To begin, gather together all equipment needed for the injection procedure. In addition, prepare 1%Evans blue dye in PBS and one insulin syringe with a 30 gauge needle per animal to be injected.
Next, remove the dam to a separate cage while manipulating the pups. When ready. Place a single pup directly on wet ice for 30 to 60 seconds.
To anesthetize. Do not leave the animal on ice too long due to the risk of hypothermia related complications while the animal is on ice. Lotus syringe with 30 microliters of Evan's blue dye.
When the animal is fully anesthetized, confirmed by lack of movement on the ice while still breathing, move it under the microscope for a right-handed injection, place the animal's muzzle to the right place. The left index finger on the muzzle and the left middle finger coddled to the earbud so that the earbud is between the index and middle fingers. Examine just anterior to the earbud for a superficial capillary that moves when the skin is manipulated.
This capillary is not the target. However, identification is important for temporal vein identification. Next, locate a dark shadowy vein inferior to the capillary that remains fixed regardless of skin position.
The temporal vein appears shadowy, runs dorsal to ventral and feeds into the jugular vein. Enter the temporal vein with the needle bevel up. If correctly inserted.
It is possible to view the needle bevel fill with blood through the skin. Then depress the plunger slowly, and note blanching of the vein down the side of the face. Allow the needle to remain within the vein for an added 10 to 15 seconds to prevent backflow of the inject.
After a proper injection, the pup should turn blue. Almost immediately. Remove the needle and use gentle force to apply a cotton swab to the injection site until the blood clots.
Allow the pup two to three minutes to recover and rewarm. Confirm that the pup is conscious, upright, and moving. Before returning to the cage, the pups can be cupped in the investigator's gloved hands to provide appropriate warmth to aid in recovery.
Alternatively, place a heating pad under the home cage to facilitate warming the injected pup. Place the pup back into the home cage and cover with bedding. To ensure reac acceptance by the dam, use a new syringe and cotton swab for each pup to maintain sterility following neonate intravenous injection of a A V nine Inod GFP transgene expression is seen throughout the brain, including the anterior striatum, as well as the pons and cerebellum.
GFP expression is also detected in the spinal cord and is seen in both spinal motor neurons and dorsal root ganglia neurons. GFP immunofluorescence is detectable in the adult mouse, heart and liver following neonatal intravenous injection of self complimentary A A V nine GFP. By comparison, GFP expression is absent in the heart and liver of unin injected mice once mastered.
This technique should take three to five minutes per animal. After watching this video, you should have a good understanding of how to perform intravenous injections into neonate mice.
