Perfusion and Fixation Protocol for Immunogold Labelling Electron Microscopy
Adapted and Prepared by Jeannie Mui
Safety Precautions
- Personal Protective Equipment (PPE): Always wear a lab coat, gloves, and safety goggles. Use a face shield if there is a risk of splashing.
- Chemical Handling:
- Paraformaldehyde and glutaraldehyde are toxic and potentially carcinogenic. Handle them in a certified chemical fume hood.
- Sodium cacodylate contains arsenic; avoid skin contact and inhalation.
- Dispose of all chemical waste in accordance with institutional hazardous waste protocols.
- Animal Welfare:
- Ensure your institutional animal care approves all procedures and use committee (IACUC or equivalent).
- Confirm deep anesthesia before proceeding with any surgical or perfusion steps.
- Minimize animal distress and handle with care throughout the procedure.
- Sharps and Instruments:
- Use caution with surgical scissors, needles, and scalpels.
- Dispose of sharps in designated containers immediately after use.
- Ventilation:
- Perform all perfusion and fixation steps inside a chemical fume hood to avoid exposure to volatile fixatives.
- Emergency Preparedness:
- Know the location of the nearest eyewash station, safety shower, and spill kit.
- In case of chemical exposure or injury, follow your lab’s emergency response procedures.
Protocol Steps
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Prepare Fixative Solution
Mix 4% paraformaldehyde (PFA) and 0.05% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). -
Set Up Perfusion System
Prepare 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 solution should be the first fluid in the tubing. The system should allow switching between channels without removing the needle from the animal.
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Prepare Fixative Vials
Fill small glass scintillation vials with fixative and place them on ice. Keep the remaining fixative at room temperature for perfusion. -
Anesthetize the Animal
Administer the appropriate anesthetic. -
Initial Recovery
Place the anesthetized animal in a heated cage for 5–10 minutes. -
Assess Anesthesia Depth
Confirm the absence of response to tail/toe pinches and the loss of ocular reflex before proceeding. -
Position the Animal
Secure the animal in a supine position on a pinnable surface (e.g., Styrofoam) inside a chemical fume hood. Gently tape the forepaws and hind paws. -
Expose the Thoracic Cavity
- Make a midline 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 area fully.
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Open the Rib Cage
- Grasp the xiphoid cartilage with blunt forceps and lift slightly.
- 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.
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Expose the Heart
- Pin or tape the rib cage laterally using 21G needles.
- Tear open the pericardial sac with blunt forceps.
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Insert the Perfusion Needle
- Secure the beating heart with blunt forceps.
- Make a 1–2 mm incision in the left ventricle and insert a 24G × 25.4 mm animal feeding needle.
- Thread the needle into the base of the aortic arch under a dissecting microscope.
- Clamp the needle in place with a hemostat.
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Begin Perfusion
- Cut the right atrium to allow drainage.
- Immediately begin perfusion with Ringer’s lactate at 10 mL/min.
- Continue until the outflow is clear.
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Switch to Fixative
- Transition to the fixative solution.
- Perfuse with 20–30 mL of fixative.
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Tissue Collection
- Dissect the tissue of interest, and immediately transfer to glass vials containing fixative (4% PFA and 0.05% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4).
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Sample Preparation for EM.
- Store at 4 °C and bring to the FEMR within four hours for LR White embedding and UV (cold) polymerization.