3.

1 FIXATION BY PERFUSION
THIS METHOD IS TO BE APPLIED WHEN PRESENTED WITH
NEUROLOGICALLY AFFECTED MICE.
Fixation by perfusion is currently performed with 4% buffered
Paraformaldehyde or periodate-lysine-2% Paraformaldehyde for samples
intended for Immunohistochemistry at the light microscopic level.
Some organs may require a brief pre-wash of the vascular system.
Vasodilating substances may prevent vascular constriction, but are in most
cases unnecessary.
Perfusion only speeds up the delivery of the fixative and not its chemical
reaction with the tissue. For this reason, perfusion-fixation is always followed
by immersion-fixation with the same fixative for a few hours.
EQUIPMENT
Perfusion Pump (or syringes may be used for smaller animals such as mice)
Perfusion catheter (0.8 mm)
Cork board
Preparation of the anaesthetic mixture:
Mix 0.75 ml of Rompun, 1 ml of Imalgene and 4 ml of distilled water to produce the
anaesthetic mixture. Label and date the mixture and store at 4C for no longer than a
month. Dose: 0.1ml/100g animal plus additional 0.2mls.
Rompun (Xylazine, Bayer)
Imalgene 1000 (Ketamin, Merial, Virbac)
Dulbeccos phosphate buffered saline (PBS)
Perfusion needle 0.5 mm (Terumo NN-2516R)
Plastic tubes (10 and 50 ml)
PROCEDURE
Anaesthetise the mouse by intraperitoneal (between the hind legs) injection
with a lethal dose of the anaesthetic mixture.
Monitor the animal carefully to ensure that the mouse is under profound
anaesthesia (i.e. unresponsive to noxious stimuli, such as the pedal reflex).
Place the mouse supine upon a clean paper towel and pin all 4 extremities to a
cork board. Moisten the fur with 70% ethanol and using a pair of sharp
scissors make a median incision from the pubic symphysis to the xyphoid
process of the sternum in order to open the abdomen. Cut the skin and

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underlying muscles along the inferior border of the rib cage on both sides of
the midline.
Hold the inferior section of the sternum with forceps to access the thoracic
cage and cut the insertions of the diaphragm on the sternum and rib cage.
Expose the inside of the thoracic cage by cutting the ribs on both sides.
Raising the sternum separate the insertions of its inner face with the parietal
pleura and the pericardium. Care should be taken not to damage any of the
great thoracic vessels.
Recline the anterior wall of the thorax towards the head and maintain this
position with a weight (e.g., a pair of large scissors).
Check that air bubbles are not apparent in the perfusion catheter.

Set the flow of the fixative at approximately 3.5 ml/minute. Note that the
fixative must flow rapidly, but drop by drop (not continuously).
Insert the perfusion needle into the left ventricle and start the perfusion pump.

Open the right auricle immediately using a pair of sharp scissors in order to
allow replacement of the blood by the fixative.
EVALUATION OF THE PERFUSION EFFIENCY
The colour of the liver should turn from deep red to pale pink within 1 minute.
If the livers colour does not change, reposition the needle in the left ventricle
and then restart perfusion.
The tail of the mouse should become stiff and the abdominal organs must
harden.

Discontinue perfusion after approximately 4 minutes (15-20 ml of fixative

perfusion).
Fix further tissues perfused with 4% paraformaldehyde or PLP, by immersion
in 10-20 volumes of the same fixative solution at 4C for between 4-12 hours,
dependent on the size of the tissue sample.
Wash the catheter and perfusion needle with deionised water.

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