Blastocyst isolation. Diagram of the uterine horns during different stages of blastocyst isolation. (A) Appearance of the attached uterine horns immediately after removal from the mouse. At this stage fat and associated tissues can be removed easily if the uterus is placed on a clean paper towel. Note the indicated cut sites. Both horns should be cut in same locations. (B) Illustration of the flushing procedure. Insert the blunted 25-G needle into the opening while the uterus is still on the paper towel. Clamp down on the needle with forceps to pinch the tissue on the needle and move the assembly over a watch glass to begin flushing.

Blastocyst injections. Phase-contrast and Nomarski differential interference contrast micrographs of the injection procedure. When an injection pipet with a beveled tip is used, the inner cell mass should be oriented at 6 or 12 o'clock to avoid damage. (A) Single-cell suspension of ES cells. Pick up only those cells that are round and have sharp refractal borders (1). Avoid cells with rough borders and those beginning to “bleb” (2). The large cells (3) are fibroblasts and should be avoided. Pick cells that will fit into the injection tip without compression. (B) Blastocysts ready for injection. Note the large blastocoel cavities and the presence of the outer shell (zona pellucida). Blastocysts without zonas cannot be injected. (C) Blastocyst and pipets in correct orientation for injection. Note the tips of both pipets and the outer edge of the blastocyst are in the same focal plane. (D) Compressed blastocyst prior to penetration of the injection tip. Rolling of the blastocyst will be observed at this stage if the tips are not properly aligned. When using beveled injection tips (as compared to the blunt tip in the photograph) the blastocysts cannot be compressed to the same degree without penetration. (E) Ejection of ES cells into the blastocoel cavity. (F) Blastocysts at different stages of collapse and re-expansion. The blastocoel cavity will collapse almost immediately after injection and appear like a ball of cells as seen in the upper part of the photograph. Over time, most embryos will re-expand and display a characteristic blastocoel cavity.

Reimplantation. Diagrams of various aspects of the reimplantation procedure. (A) A transfer pipet loaded for reimplantation. Air bubbles surround the blastocyst and act as markers that can be seen during surgery to ensure that the embryos are expelled into the uterus. Blastocysts do not have to be as tightly packed as illustrated. (B) Proper location of the skin incision for access to both uterine horns from a single site. The incision is on the midline of the back at the level of the last rib. The incision site can be slid to either side to gain access to either horn. Skin incisions can be made directly over the ovaries if the investigator prefers. Two incisions would be necessary to reimplant embryos in both horns. (C) Isolated uterine horn ready for puncture with a 25-G needle. The uterus is secured by a clamp attached to the ovarian fat pad. Blunt forceps are used to grasp the uterus near the oviduct junction as the tissue is punctured and the transfer pipet is inserted. The uterus should be held gently, to avoid damage.