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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)

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Safety Precautions

• 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.

• Chemical Safety:

  • GlutaraldehydeÌýandÌýparaformaldehydeÌýare toxic and potentially carcinogenic. Handle all fixatives in a certified chemical fume hood.
  • Sodium cacodylateÌýcontains 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.

• Animal Welfare:

  • Ensure that your institutional animal care committee approves all procedures and that the use committee (IACUC or equivalent) is involved.
  • 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.

• Ventilation and Workspace:

  • Perform all perfusion and fixation steps inside aÌýchemical fume hood.
  • Ensure the workspace is clean, organized, and clutter-free.

• 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

• PrepareÌý1 L of fixative:
  • 2.5% glutaraldehyde
  • 2.0% paraformaldehyde
  • In 0.1 M sodium cacodylate buffer, pH 7.4

2. Perfusion Setup

• Set up aÌýperistaltic pumpÌýorÌýgravity-fed IV poleÌýwith 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 requiring the removal of the needle from the animal.

3. Fixative Handling

• Fill small glass scintillation vials with fixative and place them onÌýice.
• Keep the remaining fixative atÌýroom temperatureÌýfor perfusion.

4. Anesthesia and Preparation

• Administer an appropriateÌýanestheticÌýto the mouse.
• Place the mouse in aÌýheated cageÌýfor 5–10 minutes.
• ConfirmÌýdeep anesthesiaÌýby checking for the absence of response to tail/toe pinches and loss of ocular reflexes.

5. Surgical Exposure

• Secure the mouse in aÌýsupine positionÌýon a pinnable surface (e.g., Styrofoam) inside aÌýchemical fume hood.
• Make aÌýmidline skin incisionÌýfrom just below the xiphoid process to the clavicle.
• Make twoÌýlateral incisionsÌýfrom the xiphoid process along the base of the rib cage.
• Reflect the skin flapsÌýrostrally 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 theÌýdiaphragmÌýfrom the chest wall on both sides.
• Pin or tape the rib cageÌýlaterallyÌý(e.g., using 21G needles) to expose the heart.

7. Cardiac Cannulation

• Tear open theÌýpericardial sacÌýusing blunt forceps.
• Secure theÌýbeating heartÌýand make a 1–2 mm incision in theÌýleft ventricle.
• Insert aÌý24G × 25.4 mm animal feeding needleÌý(bulbous tip to prevent damage).
• Thread the needle into theÌýaortic archÌýunder a dissecting microscope.
• Clamp the needle in place with aÌýhemostat.

8. Perfusion

• Cut theÌýright atriumÌýto allow drainage.
• Begin perfusion withÌýRinger’s lactateÌýat 10 mL/min as soon as blood flow is observed.
• Continue until the outflow isÌýclear.
• Switch to theÌýfixative solutionÌýand perfuse withÌý20–30 mLÌý(adjust for larger animals).

9. Brain Extraction and Post-Fixation

• Decapitate the mouse using large surgical scissors and remove the skin.
• Cut along theÌýmid-sagittal sutureÌýof the skull and hemisect it with a rapid, firm razor blade motion.
• Place both halves of the head intoÌý20 mL of ice-cold fixativeÌýand gently rock atÌý4 °C for 10–12 hours.

10. Tissue Dissection

• Dissect regions of interest no larger thanÌý3.0 × 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 containingÌýfixativeÌý(2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4).
• Store atÌý4 °CÌýfor no longer thanÌý1–2 weeks.
• Transport samples to theÌýFEMR (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 requiring the removal of 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)

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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 inÌý2.5% glutaraldehyde in 0.1 M phosphate buffer.
• Volume of fixative should be at least 10–20 times the volume of the tissue.
• Fix atÌý4°CÌýforÌý2–4 hoursÌý(or overnight for larger samples).
• Gently agitate if possible to ensure even penetration.

3. Rinsing

• Rinse tissue inÌý0.1 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 inÌý1% osmium tetroxideÌýin phosphate buffer for 1–2 hours at 4°C.
• Rinse again in buffer before dehydration.

5. Dehydration and Embedding

• Dehydrate through a 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.	Prime the tubing before insertion; gently tap the lines to release any trapped air.
Tissue appears under-fixed	Insufficient perfusion volume or flow rate.	Increase the fixative volume to 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.