Aldehyde Fixation Protocols

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
  • 1.0 M MgSO4
  • 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 or CB stock
    • 10 ml 25% Biological Grade GA
    • 5 ml Shandon 18.3% FA
    • 3 g sucrose
    • 0.1 ml 1 M MgSO4
    • 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


Disclaimer: As conditions of use are outside our control, we make no warranties, express or implied, and assume no liability in connection with use of this protocol.

© 2000 Robert E. Marc
This protocol may be copied and distributed for educational and non-profit
purposes as long as acknowledgement of our copyright is included
in every instance of use. It may not be sold or distributed for commercial gain.