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Immersion Fixation Protocol for Immunogold Labelling Electron Microscopy of Tissue with LR White

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 the lab’s emergency response procedures.

Procedure

1. Tissue Preparation

• Saturate area of interest with fixative and then dissect tissue into pieces no larger than 2.0 x 2.0 (cutting face) x 2.5 mm (depth) for TEM; ≤3 mm for light microscopy.
• Any additional trimming should be performed in a glass dish containing fixative to ensure the tissue remains fully submerged at all times.
• Work quickly to minimize autolysis.

2. Primary Fixation

• Immerse tissue in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer + 4.0% paraformaldehyde.
• The volume of fixative should be at least five times that of the tissue.
• Gently agitate (e.g., belly dancer), if possible, to ensure even penetration.

3.  Sample Preparation for EM

• Store at 4 °C and bring to the FEMR within four hours for LR White embedding and UV polymerization.