Pre-sterilize two pairs of fine forceps, one pair iris scissors and one blunt-ended 23 gauge needle.

Take 3.5 day pregnant mouse, kill and dissect out entire uterus using sterile technique. Place uterus in petri dish from which you will work. The uterus is trimmed free of fatty mesometrium and laid out flat in the petri dish. The ovaries, oviducts and utero-tubal junction are cut from both uterine horns and discarded. The cervical bifurcation is also removed and thus the two uterine horns have been individually isolated.

To flush the uterine horn, use a syringe of DMEM with Hepes with a blunt-ended 23 gauze needle attached. Using sterile forceps, pick up one uterine horn at one end and insert blunt-ended needle just inside the open end of the horn. Gently squeeze the plunger on the syringe and flush DMEM with hepes medium through the uterine horn into a sterile petri dish. It is important to flush gently at first otherwise the uterine contents may be lost on a wall, lab coat, etc. Turn uterine horn around and flush from other end.

Repeat with remaining uterine horn. Be sure to flush from both ends of horn. Blastocysts that were present in the uterus will have been washed out and should be in the petri dish. Collect blastocysts in a transfer pipette and transfer to small drops of M16 medium in small petri dish. Cover with 200 FLUID and incubate at 37°C with 5% CO2.

Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au       David Bowtell PMCI October 1998