Protocol for Making a 4% Formaldehyde Solution in PBS

The vast majority of IHC/ICC procedures employ fixation of tissues and cells using formaldehyde-based fixatives. The protocol below describes the technique for generating a 4% formaldehyde solution in PBS. The most effective fixative must be determined experimentally.

CAUTION:Formaldehyde is toxic. Please read the MSDS before working with this chemical. Gloves and safety glasses should be worn and solutions made inside a fume hood.

Please read the protocol in its entirety before starting.

Reagents Required

  • Deionized H2O
  • HCl (Dilute)
  • NaOH (1 N)
  • Paraformaldehyde Powder
  • 1X PBS: 0.137 M NaCl, 0.05 M NaH2PO4, pH 7.4


  • Filter Units
  • Glassware & Stir Bar (dedicated for formaldehyde solution)
  • Gloves & Eye Protection
  • Hot Plate with Magnetic Stirrer
  • Thermometer
  • Ventilated hood



  1. For 1 L of 4% Formaldehyde, add 800 mL of 1X PBS to a glass beaker on a stir plate in a ventilated hood. Heat while stirring to approximately 60 °C. Take care that the solution does not boil.
  2. Add 40 g of paraformaldehyde powder to the heated PBS solution.
  3. The powder will not immediately dissolve into solution. Slowly raise the pH by adding 1 N NaOH dropwise from a pipette until the solution clears.
  4. Once the paraformaldehyde is dissolved, the solution should be cooled and filtered.
  5. Adjust the volume of the solution to 1 L with 1X PBS.
  6. Recheck the pH, and adjust it with small amounts of dilute HCl to approximately 6.9.
  7. The solution can be aliquoted and frozen or stored at 2-8 °C for up to one month.

The Difference Between Paraformaldehyde, Formaldehyde, & Formalin

Paraformaldehyde (chemical name is polyoxymethylene) is a powder of polymerized formaldehyde that by itself cannot fix tissues. To be usable as a tissue fixative, paraformaldehyde has to be dissolved in hot water to become a formaldehyde solution. Formalin is a saturated formaldehyde solution in water (37% by weight, 40% by volume) containing 10-15% methanol. Methanol is added to slow down the polymerization to formaldehyde, which reduces the fixing power of formalin. Formalin can also be made in an alcohol-free form from powdered paraformaldehyde.

Formal 2