Adapted and Prepared by Jeannie Mui
Transcardial perfusion fixation is considered the gold standard for preserving ultrastructural detail and is generally preferred over immersion fixation. However, if immersion fixation is necessary, it is critical to immerse the tissue in fixative immediately after dissection. If additional trimming or blocking is required, perform these steps in a glass dish containing fixative to ensure the tissue remains fully submerged at all times. Failure to do so may result in tissue degradation and poor sectioning quality.
For most tissue types, optimal fixation is achieved using 2–2.5% paraformaldehyde (PFA) and 2–2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer. While phosphate-buffered fixatives can be used during perfusion, they tend to precipitate when subsequently washed with sodium cacodylate buffer and are, therefore, not recommended for immersion protocols.
The ideal concentrations of glutaraldehyde and PFA may vary depending on the tissue type, region of interest, and experimental goals.
(A) Brain Perfusion and Fixation Protocol (Mouse)
(B) Other Tissue Fixation Protocol (Mouse)
(C) Immersion Fixation Protocol Using Glutaraldehyde (Mouse)
🛡️ Safety Precautions
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Personal Protective Equipment (PPE):
- Always wear a lab coat, nitrile gloves, and safety goggles.
- Use a face shield if there is a risk of splashing during perfusion or dissection.
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Chemical Safety:
- Glutaraldehyde and paraformaldehyde are toxic and potentially carcinogenic. Handle all fixatives in a certified chemical fume hood.
- Sodium cacodylate contains arsenic and must be handled with extreme care. Avoid inhalation and skin contact.
- Dispose of all chemical waste in accordance with institutional hazardous waste protocols.
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Animal Welfare:
- Ensure your institutional animal care approves all procedures and use committee (IACUC or equivalent).
- Confirm deep anesthesia before beginning any surgical or perfusion steps.
- Minimize animal distress and handle with care throughout the procedure.
-
Sharps and Instruments:
- Use caution with surgical scissors, scalpels, and needles.
- Dispose of all sharps in designated sharps containers immediately after use.
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Ventilation and Workspace:
- Perform all perfusion and fixation steps inside a chemical fume hood.
- Ensure the workspace is clean, organized, and free of unnecessary materials.
-
Emergency Preparedness:
- Know the location of the nearest eyewash station, safety shower, and chemical spill kit.
- If you are exposed to chemicals or injure yourself, follow your lab’s emergency response procedures and seek medical attention if necessary.
(A) Brain Perfusion and Fixation Protocol (Mouse)
1. Fixative Preparation
- Prepare 1 L of fixative:
- 2.5% glutaraldehyde
- 2.0% paraformaldehyde
- In 0.1 M sodium cacodylate buffer, pH 7.4
2. Perfusion Setup
- Set up a peristaltic pump or gravity-fed IV pole with two channels:
- Channel 1: Ringer’s lactate solution
- Channel 2: Fixative solution
- Ensure both lines are free of air bubbles. The Ringer’s lactate should be the first solution in the tubing. The system must allow switching between solutions without removing the needle from the animal.
3. Fixative Handling
- Fill small glass scintillation vials with fixative and place them on ice.
- Keep the remaining fixative at room temperature for perfusion.
4. Anesthesia and Preparation
- Administer an appropriate anesthetic to the mouse.
- Place the mouse in a heated cage for 5–10 minutes.
- Confirm deep anesthesia by checking for the absence of response to tail/toe pinches and loss of ocular reflexes.
5. Surgical Exposure
- Secure the mouse in a supine position on a pinnable surface (e.g., Styrofoam) inside a chemical fume hood.
- Make a midline skin incision from just below the xiphoid process to the clavicle.
- Make two lateral incisions from the xiphoid process along the base of the rib cage.
- Reflect the skin flaps rostrally and laterally to expose the thoracic cavity fully.
6. Thoracic Access
- Grasp the xiphoid cartilage with blunt forceps and lift gently.
- Insert pointed scissors and cut through the thoracic musculature and rib cage up to the clavicles.
- Detach the diaphragm from the chest wall on both sides.
- Pin or tape the rib cage laterally (e.g., using 21G needles) to expose the heart.
7. Cardiac Cannulation
- Tear open the pericardial sac using blunt forceps.
- Secure the beating heart and make a 1–2 mm incision in the left ventricle.
- Insert a 24G × 25.4 mm animal feeding needle (bulbous tip to prevent damage).
- Thread the needle into the aortic arch under a dissecting microscope.
- Clamp the needle in place with a hemostat.
8. Perfusion
- Cut the right atrium to allow drainage.
- Begin perfusion with Ringer’s lactate at 10 mL/min as soon as blood flow is observed.
- Continue until the outflow is clear.
- Switch to the fixative solution and perfuse with 20–30 mL (adjust for larger animals).
9. Brain Extraction and Post-Fixation
- Decapitate the mouse using large surgical scissors and remove the skin.
- Cut along the mid-sagittal suture of the skull and hemisect it with a rapid, firm razor blade motion.
- Place both halves of the head into 20 mL of ice-cold fixative and gently rock at 4 °C for 10–12 hours.
10. Tissue Dissection
- Dissect regions of interest no larger than 3.0 × 3.0 mm (cutting face) × 3.5 mm (depth).
- These dimensions are suitable for locating the region of interest using light microscopy (LM) before sectioning it for electron microscopy (EM).
11. Storage and Transport
- Place dissected tissue into glass vials containing fixative (2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4).
- Store at 4 °C for no longer than 1–2 weeks.
- Transport samples to FEMR (Room B4, Strathcona Anatomy & Dentistry Building) for EM processing (EPON).
(B) Other Tissue Fixation Protocol (Mouse)
1. Fixative Preparation
- Prepare 1 L of fixative:
- 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4
2. Perfusion System Setup
- Set up a peristaltic pump or gravity-fed IV pole with two channels:
- Channel 1: Ringer’s lactate solution
- Channel 2: Fixative solution
- Ensure both lines are free of air bubbles. The Ringer’s lactate should be the first solution in the tubing. The system must allow switching between solutions without removing the needle from the animal.
3. Fixative Handling
- Fill small glass scintillation vials with fixative and place them on ice.
- Keep the remaining fixative at room temperature for perfusion.
4. Anesthesia and Preparation
- Administer an appropriate anesthetic to the mouse.
- Place the mouse in a heated cage for 5–10 minutes.
- Confirm deep anesthesia by checking for the absence of response to tail/toe pinches and loss of ocular reflexes.
5. Surgical Exposure
- Secure the mouse in a supine position on a pinnable surface (e.g., Styrofoam) inside a chemical fume hood.
- Make a midline skin incision from just below the xiphoid process to the clavicle.
- Make two lateral incisions from the xiphoid process along the base of the rib cage.
- Reflect the skin flaps rostrally and laterally to expose the thoracic cavity fully.
6. Thoracic Access
- Grasp the xiphoid cartilage with blunt forceps and lift gently.
- Insert pointed scissors and cut through the thoracic musculature and rib cage up to the clavicles.
- Detach the diaphragm from the chest wall on both sides.
- Pin or tape the rib cage laterally (e.g., using 21G needles) to expose the heart.
7. Cardiac Cannulation
- Tear open the pericardial sac using blunt forceps.
- Secure the beating heart and make a 1–2 mm incision in the left ventricle.
- Insert a 24G × 25.4 mm animal feeding needle (bulbous tip to prevent damage).
- Thread the needle into the aortic arch under a dissecting microscope.
- Clamp the needle in place with a hemostat.
8. Perfusion
- Cut the right atrium to allow drainage.
- Begin perfusion with Ringer’s lactate at 10 mL/min as soon as blood flow is observed.
- Continue until the outflow is clear.
- Switch to the fixative solution and perfuse with 20–30 mL (adjust for larger animals).
9. Tissue Dissection and Storage
- Dissect regions of interest no larger than 3.0 × 3.0 mm (cutting face) × 3.5 mm (depth).
- These dimensions are suitable for locating the region of interest using light microscopy (LM) before sectioning it for electron microscopy (EM).
11. Storage and Transport
- Place dissected tissue into glass vials containing fixative (2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4).
- Store at 4 °C for no longer than 1–2 weeks.
- If stored for longer than two weeks, transfer the samples to a washing buffer and change it once per week.
- Transport samples to FEMR (Room B4, Strathcona Anatomy & Dentistry Building) for EM processing (EPON).
(C) Immersion Fixation Protocol Using Glutaraldehyde
Materials Needed
- 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2–7.4)
- 0.1 M phosphate buffer (for rinsing)
- Tissue samples (≤1 mm thick for TEM; ≤5 mm for light microscopy)
- Glass vials or plastic containers
- Forceps and scalpel
- Cold storage (4°C)
Procedure
1. Tissue Preparation
- Dissect tissue into small pieces (1 mm³ for TEM; up to 5 mm for light microscopy).
- Work quickly to minimize autolysis.
2. Primary Fixation
- Immerse tissue in 2.5% glutaraldehyde in 0.1 M phosphate buffer.
- Volume of fixative should be at least 10–20 times the volume of the tissue.
- Fix at 4°C for 2–4 hours (or overnight for larger samples).
- Gently agitate if possible to ensure even penetration.
3. Rinsing
- Rinse tissue in 0.1 M phosphate buffer, 3 times for 10 minutes each at 4°C.
- This removes excess glutaraldehyde and prepares tissue for post-fixation or storage.
4. Post-Fixation (Optional for TEM)
- Post-fix in 1% osmium tetroxide in phosphate buffer for 1–2 hours at 4°C.
- Rinse again in buffer before dehydration.
5. Dehydration and Embedding
- Dehydrate through graded ethanol series (30%, 50%, 70%, 90%, 100%).
- Embed in resin (e.g., Epon or Spurr) for TEM or paraffin for light microscopy.
Troubleshooting Tips
| Issue | Possible Cause | Solution |
|---|---|---|
| No fluid flow during perfusion | Blocked needle or improper insertion. | Reposition the needle; ensure it is properly threaded into the aortic arch. |
| Air bubbles in tubing | Incomplete priming of lines. | Reprime the tubing before insertion; tap lines gently to release trapped air. |
| Tissue appears under-fixed | Insufficient perfusion volume or flow rate. | Increase fixative volume; ensure consistent flow at 10 mL/min. |
| Fixative leaks from the heart | The needle is not clamped securely or inserted too shallowly. | Reinsert and secure the needle with a hemostat above the incision site. |
| Animal responds to stimuli after anesthesia | Inadequate anesthesia depth. | Wait longer post-injection; confirm with multiple reflex checks before proceeding. |
| Tissue too large for EM processing | Improper dissection dimensions. | Use a ruler or template to ensure tissue is ≤ 3.0 × 3.0 × 3.5 mm. |
| Fixative crystallization or cloudiness | Improper storage or expired reagents. | Prepare fresh fixative; store at the correct temperature and pH. |