Adapted and Prepared by Jeannie Mui
Transcardial perfusion fixation is the gold standard for fixation as it preserves ultrastructural detail and is preferable to immersion fixation. If you must perform immersion fixation, immediate immersion in fixative is essential. If the dissection requires extra time, finish trimming or blocking the tissue in a glass dish so the tissue is (always) submerged in fixative; otherwise, the tissue will fall apart during sectioning. Most types of tissues are best fixed with 2-2.5% paraformaldehyde and 2-2.5% glutaraldehyde in a 0.1M sodium cacodylate buffer. Phosphate buffer fixatives tend to precipitate out during washing with the sodium cacodylate buffer but can be used during perfusion without a problem. The percentages of glutaraldehyde and PFA vary depending on the tissue type, area of interest, etc.
(1) Brain
(2) Other Tissue
(1) Brain
- Prepare 1L fixative (2.5% glutaraldehyde, 2.0% paraformaldehyde in 0.1M sodium cacodylate buffer at pH 7.4).
- Prepare the peristaltic pump (or gravity I.V. pole) so that two tubes/channels are filled with (1) Ringer's lactate solution and (2) fixative – you should be able to switch from one channel to the other without having to remove the needle from the animal.
- Fill small glass scintillation vials with the fixative and place them on ice. Keep the rest of the fixative at room temperature for perfusion use.
- Administer anesthetic to the mouse.
- Place the injected mouse in a heated cage for 5 to 10 minutes.
- Assess responses to tail/toe pinches and observe ocular reflexes. Proceed after the mouse is unresponsive to the noxious stimuli and the reflex is absent.
- Secure the mouse in the supine position (lying on the back with face upward) by gently taping the forepaws and hind paws to a pinnable (i.e., Styrofoam) work surface inside a chemical fume hood.
- Make an incision through the skin with surgical scissors along the thoracic midline from just beneath the xiphoid process to the clavicle. Make two additional skin incisions from the xiphoid process along the base of the ventral rib cage laterally.
- Gently reflect the two flaps of skin rostrally and laterally, exposing the thoracic field completely.
- Grasp the cartilage of the xiphoid process with blunt forceps and raise it slightly to insert pointed scissors. Cut through the thoracic musculature and ribcage between the breastbone and medial rib insertion points and extend the incision rostrally to the level of the clavicles.
- Separate the diaphragm from the chest wall on both sides with scissor cuts.
- Tape or pin (e.g. 21G needles) the reflected rib cage laterally to expose the heart (and other thoracic organs).
- Gently grasp the pericardial sac with blunt forceps and tear it fully.
- Secure the beating heart with blunt forceps and make a 1-2 mm incision in the left ventricle. Immediately insert a 24G X 25.4mm animal feeding needle. The manufacturer calls this a needle, but the tip is bulbous and will not damage the heart. Thread the feeding needle into the base of the aortic arch using a dissecting microscope. Using a hemostat, clamp the needle base to the left ventricle above the incision site.
- Immediately cut the right atrium with scissors and begin the infusion of Ringer's lactate solution at 10 mL/min at the first sign of blood flow.
- Continue perfusing the body until the fluid exiting the right atrium is clear.
- Switch from saline perfusate to fixative.
- For mice, perfuse 20 to 30 mL of fixative (larger animals may require more fixative).
- Decapitate the mouse with large surgical scissors. Remove the skin.
- Cut the skull along the mid-sagittal suture. Completely hemisect the skull with a rapid, forceful razor blade thrust.
- Place the two halves of the mouse head into 20 mL of ice-cold fixative and gently rock at 4 °C for 10 to 12 hrs.
- Dissect regions of interest with dimensions no larger than 3.0 mm x 3.0 mm (cutting face) x 3.5 mm (depth; for LM sections to be stained with toluidine blue sections for selecting regions of interest for EM sections).
- Place dissected tissue into glass vials containing fixative (2.5% glutaraldehyde, 2.0% paraformaldehyde in 0.1M sodium cacodylate buffer at pH 7.4) and store at 4 °C for no longer than one to two weeks.
- Bring to the FEMR for EM processing, Room B4, in the basement of the Strathcona Anatomy & Dentistry Building.
(2) Other Tissue
- Prepare 1L fixative (2.5% glutaraldehyde in 0.1M sodium cacodylate buffer at pH 7.4).
- Prepare the peristaltic pump (or gravity I.V. pole) so that two tubes/channels are filled with (1) Ringer's lactate solution and (2) fixative – you should be able to switch from one channel to the other without having to remove the needle from the animal.
- Fill small glass scintillation vials with the fixative and place them on ice. Keep the rest of the fixative at room temperature for perfusion use.
- Administer anesthetic to the mouse.
- Place the injected mouse in a heated cage for 5 to 10 minutes.
- Assess responses to tail/toe pinches and observe ocular reflexes. Proceed after the mouse is unresponsive to the noxious stimuli and the reflex is absent.
- Secure the mouse in the supine position (lying on the back with face upward) by gently taping the forepaws and hind paws to a pinnable (i.e., Styrofoam) work surface inside a chemical fume hood.
- Make an incision through the skin with surgical scissors along the thoracic midline from just beneath the xiphoid process to the clavicle. Make two additional skin incisions from the xiphoid process along the base of the ventral rib cage laterally.
- Gently reflect the two flaps of skin rostrally and laterally, exposing the thoracic field completely.
- Grasp the cartilage of the xiphoid process with blunt forceps and raise it slightly to insert pointed scissors. Cut through the thoracic musculature and ribcage between the breastbone and medial rib insertion points and extend the incision rostrally to the level of the clavicles.
- Separate the diaphragm from the chest wall on both sides with scissor cuts.
- Tape or pin (e.g. 21G needles) the reflected rib cage laterally to expose the heart (and other thoracic organs).
- Gently grasp the pericardial sac with blunt forceps and tear it fully.
- Secure the beating heart with blunt forceps and make a 1-2 mm incision in the left ventricle. Immediately insert a 24G X 25.4mm animal feeding needle. The manufacturer calls this a needle, but the tip is bulbous and will not damage the heart. Thread the feeding needle into the base of the aortic arch using a dissecting microscope. Using a hemostat, clamp the needle base to the left ventricle above the incision site.
- Immediately cut the right atrium with scissors and begin the infusion of Ringer's lactate solution at 10 mL/min at the first sign of blood flow.
- Continue perfusing the body until the fluid exiting the right atrium is clear.
- Switch from saline perfusate to fixative.
- For mice, perfuse 20 to 30 mL of fixative (larger animals may require more fixative).
- Dissect the region of interest with dimensions no larger than 3.0 mm x 3.0 mm (cutting face) x 3.5 mm (depth; for LM sections to be stained with toluidine blue sections for selecting regions of interest for EM sections).
- Place the tissue into glass vials containing fixative (2.5% glutaraldehyde in 0.1M sodium cacodylate buffer at pH 7.4) and store at 4 °C for no longer than one to two weeks.
- Bring to the FEMR for processing for EM, Room B4, in the basement of the Strathcona Anatomy & Dentistry Building.