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
- PBS (1x): 0.137 M NaCl, 0.05 M NaH2PO4, pH 7.4
Materials
- Filter units
- Glassware and stir bar (dedicated for formaldehyde solution)
- Gloves and eye protection
- Hot plate with magnetic stirrer
- Thermometer
- Ventilated hood
Procedure
- 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 pipet 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 4 °C for no longer than 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.