Fixation by perfusion
|Affiliation||Department of Cell Biology, Graduate School of Medicine, Osaka University|
|Contact||inquiry about this protocol|
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- Pour hot water into styrofoam and dip a beaker with Rat ringer and a beaker with fixative into the hot water to warm up to 38-42℃
- Put a 23G needle to the tip of plastic tube of perfusion pump and seal the junction with Parafilm. Fit in the tube to a perfusion pump. Fix the tube firmly to the perfusion pump with a packing tape.
- anesthetize mice with an anesthetic agent (‘Nembutal’) intraperitoneally and fix the mice onto the dissection table by pinning their limbs with needles. * the amount of anesthetic agent (‘Nembutal’) is 0.01-0.02ml of 1/10 diluted Nembutal per 1g body wt
- open the abdomen with scissors and tweezers and next open the thorax, but if you open the thorax, mice cannot breath, so after opening the thorax, you have to do the following procedures as fast as possible.
- open the thorax by cutting the diaphragm along the thoracic border (perpendicular to the long axis of the body) and then cut right and left part of the rib cage along the long axis of the body to make one easy to look at the heart. Turn over the central part of rib cage (with sternal bone) and pin it down by a long needle (21G) to a dissection table
- inject diluted heparin (-0.05cc) to the left ventricule (the apex of the heart).
- After inserting the needle of the perfusion pump into the left ventricule, cut a part of the right atrium (which is located in the right deep part of the heart) with an ophthalmologist scissor. Soon after the scission, turn on the perfusion pump to infuse the Ringer’s solution. * take care that the light should be bright and properly directed enough to identify the right atrium which is located at the deep part of the heart * the flux of the Ringer has to be slow at the beginning (scale 2-3) and then speed up to scale 5 (younger than 14d) or scale 7- (14d or older).
- When the outflow from the atrium turns transparent, turn off the pump completely and then move the other tip of the perfusion tube from the Ringer to fixative and then restart the pump immediately and speed it up rapidly to full extent (depending on the age). * take extreme care not to include air into the perfusion tube because air clogging in the tissues prevent effective fixation, so stop the pump completely before moving the tip of the tube out from the Ringer.
- Perfuse the mouse with 50ml fixative (adult, less is sufficient for youner mice)