Post #4: Perfusions

One needs to preserve brain tissue and cells in order to see neurons. To do this, we perfuse the mouse. A perfusion runs preservative solution through the mouse in order to keep the integrity of the brain cells as the tissue undergoes sectioning and immunocytochemistry. We run a solution called Zamboni’s fixative through the mouse to preserve tissue.  Although commonly  used for electron microscopy, Zamboni’s fixative is  a good general purpose fixative useful in preserving the brain tissue that we are examining. Zamboni, as we call it, is a phosphate buffered combination of paraformaldehyde and picric acid. It stabilizes cellular proteins without being destroyed by tissue fluids.

Perfusions are done between one to two weeks after a mouse has been trapped. The mouse is put into isoflurane, an inhaled anesthetic, after the mouse becomes unconscious, the mouse is weighed. The mouse is given more isoflurane until the mouse is euthanized. Once breathing stops, the chest cavity is opened to expose the heart. A needle is placed into the left ventricle. This needle is connected to a pipe that leads to  a container of Phosphate buffer. A pump runs buffer through the mouse to remove the blood from the mouse’s system. Surgery needs to be done quickly so that the blood doesn’t clot in the mouse and prevent solution from running through the circulatory system. An indicator of sucessfull flow of phosphate buffer is the lightening of the liver from a dark reddish brown to a pale tan. Once all the blood has been taken out of the mouse, the solution is changed into Zamboni. If done right, the fixative should reach all parts of the body. The mouse’s toes and mouth should turn yellow from the fixative and the tail should become stiff. Good circulation will most importantly be needed for the brain to be preserved for the next stages of the experiment.

One the mouse has ben perfused, two things happen: the brain is taken out and the gonads are removed. The brain is obtained by opening the top of the skull. The brain is placed in -4 degrees in Zamboni for one night on a shaker, a 15% sucrose solution for one night on a shaker, and then 30% sucrose solution until sectioning can be done. The gonads are also taken from the animal. For females, the ovaries and uterine horn are weighed separately. For males, paired testes weight and seminal vesicle weight is recorded. The weights of these reproductive organs will indicate reproductive status of the mice.