Immersion Fixation Methods for Mammalian Tissues

Stock materials

• Paraformaldehyde (PF) powder
• 18.3% formaldehyde (FA) solution (Shandon)
• 25% Biological grade glutaraldehyde (GA) (Fisher)
• 25% EM grade GA (Sigma-Aldrich)
• 0.2 M phosphate buffer pH 7.4 (PB)
• 0.2 M cacodylate buffer pH 7.4 (CB)
• 0.2 M NaOH
• 0.02% CaCl2 dihydrate
• sucrose
• deionized water (dH2O)
• Magnetic stirrer/hotplate

Fixative Recipes

• Fixative 1: routine 1% FA / 2.5% GA fixative for HPI; prepare fresh
• 50 ml cc 0.2 M PB stock
• 10 ml 25% Biological Grade GA
• 5 ml Shandon 18.3% FA
• 3 g sucrose
• dilute to 100 ml with dH2O
• Fixative 2: 1% FA / 2.5% GA for electron microscopy- prepare fresh
• 10% PF stock (prepare in fume hood only!)
  1. 4 drops 0.2 N NaOH in 10 ml dH2O in a glass vial
  2. 1 g PF
  3. Bring to near boiling in fume hood with stirring
  4. Stop when clear, cool to room temperature and filter
• 50 ml full fixative
1. 10 ml dH2O
2. 5 ml 10% PF (from above)
3. 5 ml 25% EM grade GA
4. Buffer options: 25 ml 0.2 M CB or PB
5. 1.5 g sucrose
6. 3 ml 0.02% CaCl2
7. Dilute to 50 ml with dH2O
8. Check pH (7.4) and adjust if necessary
• Fixative 3: 4% FA / X % GA for macromolecule immunocytochemistry; prepare fresh
• 20% PF stock (prepare in fume hood only!)
1. 8 drops 0.2 N NaOH in 10 ml dH2O in glass vial
2. 2 g PF
3. Bring to near boiling in fume hood with stirring
4. Stop when clear, cool to room temperature and filter
• 50 ml full fixative
  1. 10 ml dH2O
  2. 10 ml 20% PF (from above)
  3. Buffer options: 25 ml 0.2 M CB or PB
  4. 1.5 g sucrose
  5. 3 ml 0.02% CaCl2
  6. Dilute to 50 ml with dH2O
  7. Check pH (7.4) and adjust if necessary
  8. Add X microliters 25% Biological Grade GA for the following concentrations
  • 0.1% GA: add 200 microliters of 25% GA
  • 0.05% GA: add 100 microliters of 25% GA
  • 0.02% GA: add 40 microliters of 25% GA
  • 0.01% GA: add 10 microliters of 25% GA