4. Make midline incision in skin from xyphoid process to pubis using scalpel, and retract; attaching retractors firmly to styrofoam support will create a stable working field. Stop any bleeding with cotton swabs before carefully retracting fascia and peritoneum, and incising them in the midline with scissors (care is required here not to incise underlying bowel). Continue incision cephalad along midline of fascia (where there are few blood vessels) to expose entire abdominal contents. If necessary to expose the uterus, gently pack the abdomen with cotton balls or swabs to remove the intestines from the opertive field, being careful not to lacerate or obstruct the bowel. Fill the peritoneal cavity with LR, and lavage until clear if the solution turns at all turbid.
Wide exposure is important to allow the later manipulations. During the remainder of the operation, keep the peritoneal cavity moist and free of blood; dehydration or blood around the uterus increases the rate of post-operative abortion.
5. Elevate the embryos one at a time out of the peritoneal cavity, and transilluminate with a fiberoptic light source to visualize the structure to be injected. For lateral cerebral ventricular injections, for example, the cerebral venous sinuses serve as landmarks.
When deciding on a structure to inject, keep in mind that free diffusion of virus solution throughout a fluid-filled structure lined with mitotic cells is best for ensuring even distribution of viral infection events throughout the tissue being labelled. The neural tube is an example of such a structure; when virus is injected into one lateral ventricle, it is observed to quickly diffuse throughout the entire ventricular system.
6. Using a heat-drawn glass micropipette attached to an automatic microinjector, penetrate the uterine wall, extraembryonic membranes, and the structure to be infected in one rapid thrust; this minimizes trauma and improves survival. Once the pipette is in place, inject the desired volume of virus solution, usually 0.1-1.0 ml. Co-injection of a dye such as 0.005% (wt/vol) trypan blue or 0.025% fast green helps determination of the accuracy of injection and does not appear to impair viral infectivity; coinjection of the polycation polybrene (80 mg/ml) aids in viral attachment to the cells to be infected. The type of instrument used to deliver the virus depends on the age of the animal and the tissue to be injected. At early embryonic stages, the small size and easy penetrability of the tissue makes a pneumatic microinjector (such as the Eppendorf 5242) best for delivering a constant amount of virus at a controlled rate with a minimum of trauma. Glass micropipettes should be made empirically to produce a bore size which will allow penetration of the uterine wall and the tissue to be infected. At later ages, (late embryonic and postnatal) a Hamilton syringe with a 33-gauge needle works best.
