1. Place a paper towel in the gas chamber.
2. Pick up the mouse with its tail and place it in the gas chamber.
3. Attach tube to gas tank and turn on till mouse dies.
4. Then put the mouse in a beaker containing soap detergent and wash by dipping mouse into and out of the beaker.
5. Wash with distilled water and place the mouse ventrally on another paper towel on the counter.
6. Lift the skin near stomach with forceps and cut skin as shown.
7. Dissect out the uterine sac and wash with distilled water to remove blood.
8. Under microscope remove white membrane, remove placenta by cutting umbilical cord close to stomach.
9. Place the uterine sac in a Petri dish which contains 1xPBS solution.
10. Measure the embryo (cephalic to caudal end) with a mm ruler to determine age.
11. Amputate head of the embryo.
12. Carefully open the chest cavity to expose the heart. Be careful not to damage the heart.
13. Submerge the embryo in prewarmed 1X PBS containing KCl (37 degrees; 1 ml of 3M KCL in 50 mL 1x PBS without Ca/Mg).
14. Place on shaker for 15 minutes to allow perfusion . Put embryo into 10% phosphate buffered formalin in PBS. Minimum fixation times are as follows:

Age	Minimum Fixation Time
E9.5	12 hours
E10.5 to E13.5	1 day
E14.5 to E16.5	2 days
E17.5	3 days
E18.5 to Neonates	5 days

Embryos/fetuses can be left in fixative for many weeks and still provide good EFIC imaging. The fixation time is minimum time required in fixative before the samples can be processed for paraffin/PEG embedding.