This is a cached page for the URL ( To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Protocol for Making a 4% Formaldehyde Solution in PBS
Technical Information

Technical Information

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 and stir bar (dedicated for formaldehyde solution)
  • Gloves and 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, and 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.