HISTOTECHNOLOGY AND COMPARATIVE PATHOLOGY FACILITY

Procedures & Optimal Fixation
Ideally, animals should be euthanized and tissues placed immediately into appropriate fixative

If you cannot necropsy the animal immediately: 

  • At the very least, put it at 4C (DO NOT FREEZE)
  • Preferably place entire animal in fixative (flat bottomed container)
  • Open abdominal & thoracic viscera and snip skull
  • fix (± gently inflating lungs and GI with fix)
  • Single small incision in kidneys and liver if possible

Fixation methods 

  • Perfusion fixation followed by immersion fixation
  • Immersion fixation (do not re-use fixative)
    • Ideal fixative-tissue ratio 10:1—thus the mouse in a 50 mL conical is not ideal (use a flat bottomed container)
    • Tissues should be no thicker than 5 mm for optimal fixation
    • Bones should be cracked to allow for fixation of marrow
    • Infusion of nasal cavities for optimum fixation of nasal structures
    • Inflate lungs and intestinal tract with fixative
    • If eyes are primary interest, need to identify appropriate fixative for your interests
    • If testes are primary interest, consider Bouin’s
Fixative options

10% Neutral Buffered Formalin

Zinc Formalin

4% Paraformaldehyde

Bouin's Solution

Frozen tissues (Frozens)

 
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