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Fixation Protocols

Immersion fixation


Place the mouse in dorsal recumbancy and open the ventral midline from pubis to the base of the neck. Split the mandible to remove the tongue, trachea, heart and lungs. Inflate the lungs with fixative. Remove the abdominal viscera. Turn the mouse over and remove the skin of the head. Decapitite the animal and remove the skull cap to expose the brain, which is left in situ. Place the organs and the remainder of the body in fixative so that tissues are completely covered. A ratio of 1:10 tissue: fixative is optimal.
Recommended fixatives: 10% formalin or 4% paraformaldehyde for most purposes, Feketes Acid Alcohol for nervous tissue, Bouin’s solution for reproductive tissue. Except for Bouin’s solution, tissues may be stored indefinitely, however antigenicity may be lost for subsequent immunohistochemical purposes.

Neonates and embryos

Remove the skin from the head, and perform a single midline ventral incision to open the abdominal and thoracic cavities before placing the mouse in fixative.
Recommended fixatives: Bouin’s solution. Particularly with Bouin’s solution, the tissue will become brittle if placed in fixative for too long. Embryos with a crown-rump length of 2mm require only 1 hour in fixative, while those with a crown-rump length of 15 mm can be placed in fixative for up to 24 hours. After removal from fixative, embryos may be placed in 70% ethanol for long-term storage at room temperature.

Perfusion fixation

Fixation by intracardiac perfusion is recommended for fixation of tissues which autolyse rapidly, such as nervous tissue or endocrine tissue. The mouse is anesthetized with an intrperitoneal injection of Avertin, or Ketamine/Xylazine. Once deep anesthesia is attained (absence of withdrawel reflex when the foot is firmly pinched with forceps), the mouse is pinned in dorsal recumbancy. The chest is opened, and the right atrium removed with scissors. A 21ga butterfly needle is placed in the left ventricle, and 5-20 ml of saline flushed in over the course of about a minute. Thereafter, 30-50 ml of fixative is flushed in until the body becomes stiff. The skull is then opened, and the abdomen opened along the midline before the mouse is immersed in fixative for another 7-10 days.

Gentle fixation for tissue arrays

Place tissues in 4% paraformaldehyde for no longer than 48 hours at 4oC. The solution should completely cover the tissue. After 24-48 hours, tissue can then be stored in 1X PBS at 4oC for up to two weeks. Tissues may also be stored in 70% ethanol at 4oC.