Prior to starting the murine embryo isolation, clean the area thoroughly with 70%ethanol. Ensure all dissection tools are washed and sterilized. Obtain five milliliters of sterile DMEM containing 10%FBS, and 20 milliliters of DPBS and place them on ice.
Next to perform the embryo isolation, using a stereo microscope with a transmitted light stage and camera, place the properly euthanized pregnant dam on its back and sterilize the area near the vaginal opening with ethanol. Using dissection scissors, and tweezers, lift the skinfold near the vaginal opening and make a small V-shaped cut slowly revealing the uterus of the pregnant dam. Then dissect out the uterine horn of the dam by holding one end of it with tweezers and cutting along it.
Making sure to remove the cervix. Place the uterine horn into a 10 centimeter Petri dish containing DPBS on ice. With dissection scissors, and tweezers, remove the uterine muscle along the implantation sites and cut each implantation site or pearls containing the decidual swellings inside.
Place it into fresh DPBS in a six centimeter Petri dish on ice. Take one implantation site. Place it in a new six centimeter Petri dish on top of the stereo microscope stage, and add 500 microliters of DPBS to it.
Adjust the focus of the microscope and the light source. Hold down the implantation site with one set of tweezers in one hand, and with the other hand slowly insert another pair of forceps into the end of the implantation site cut from the uterine horn, gradually revealing the deciduous swelling. To reveal the embryo, hold the anti mistrial end of the isolated decidual swelling with one pair of forceps and with the other pair, slowly make a horizontal cut about one quarter of the size of the decidual swelling from the mistrial end.
Now with both forceps slowly push from the anti mistrial end of the decidual swelling until the embryo pops out from the freshly cut mistrial end. Once the embryo is revealed, proceed to remove any extra embryonic tissues attached. If the parietal endodermal sac and ecto placental cone do not spontaneously come off the embryo, use a pair of forceps to remove them along with any associated maternal blood.
Then while holding down the embryo with one pair of forceps, slowly peel the visceral yolk sack from the embryo using another pair of forceps. Locate the parietal endodermal sac and ectoplacental cone. Using AP 20 pipette, transfer the yolk sac with no more than 10 microliters of DPBS from the dish into an eight strip PCR tube on ice.
Take bright field pictures of freshly isolated embryos to ensure the staging of the litter mates is similar. With a P200 pipette slowly transfer the embryo with 50 microliters of DMEM containing 10%FBS into a 1.5 milliliter tube kept on ice. Repeat the same for all decidual swelling, using clean tools and new plastics for each isolation.
