Figure 9

Practical points to improve accuracy. (A) Diagram of the optimum experimental set-up enlarged from that shown in figure 8. The uterine stabilizer (1) is cast in one of the modified Petri dishes so that it extends into the central hole (2). As a result the uterine stabilizer fits more closely to the transparent membrane (3) attached to the under surface of the petri dish and also creates a more secure seal with the skin of the mouse abdomen (4). This is important because it reduces the possibility of the exposed segment of uterine horn slipping between the under surface of the stabilizer and the transparent rubber membrane or the maternal skin during microinjections. Keeping the exposed uterus closely approximated to the uterine stabilizer (dotted oval labelled 5 in A &C) is very important in improving accuracy of microinjections. This improves stability of the uterine segment during microinjection allowing easier penetration of the micropipette through the thick uterine muscle enabling a more accurate placement of the tip of the microinjection pipette into the target region. (B) If the uterus is separated from the edge of the uterine stabilizer (double-headed arrow), the uterus will move away from the advancing needle and the target area of the conceptus will move out of the focal zone and/or field of view of the transducer reducing the accuracy of the microinjections. (C) The optimal position of the uterus relative to the uterine stabilizer is demonstrated. The alignment of the conceptus within the scan plane is also optimal. In this position three potential targets are easily accessible from a lateral approach thereby avoiding injury to the embryo caused by the microinjection pipette or the inadvertent deposition of fluorescent beads in other targets during needle insertion or withdrawal. (D) Placement of the microinjection pipette within the amniotic cavity (arrow). (E) Immediately after removal of the microinjection pipette, a small amount of echogenic material (fluorescent beads) can be seen within the amniotic cavity (arrow) which was not present prior to microinjection (compare with amniotic cavity in image B).