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.
1X PBS: 0.145 M NaCl, 0.0027 M KCl, 0.0081 M Na2HPO4, 0.0015 M KH2PO4, pH 7.4
Glassware and stir bar (dedicated for formaldehyde solution)
Gloves and eye protection
Hot plate with magnetic stirrer
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.
Add 40 g of paraformaldehyde powder to the heated PBS solution.
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.
Once the paraformaldehyde is dissolved, the solution should be cooled and filtered.
Adjust the volume of the solution to 1 L with 1X PBS.
Recheck the pH, and adjust it with small amounts of dilute HCl to approximately 6.9.
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.