AAT Bioquest

What is the difference between formaldehyde and paraformaldehyde in cell fixation?

Posted March 22, 2018

Can you explain the difference between paraformaldehyde, formaldehyde and formalin?


Paraformaldehyde (PFA) is a polymer of formaldehyde. Paraformaldehyde itself is not a fixing agent, and needs to be broken down into its basic building block, formaldehyde. This can be done by heating or basic conditions until it becomes solubilized. 

Formalin is the name for saturated (37%) formaldehyde solution. Beware though, some commercial formaldehyde solutions contain methanol to prevent polymerization (into paraformaldehyde). Since 100% formalin contains up to 15% of methanol as a stabilizer, it has a significant impact on cell fixation.

Methanol is a permeabilizing agent. It can interfere with the staining of membrane bound proteins, and can greatly influence staining of cytoskeletal proteins. For example, when staining cellular F-actin it is imperative to use a methanol-free formaldehyde fixative. This is because methanol can disrupt F-actin during the fixation process and prevent the binding of phalloidin conjugates.

"Pure" methanol-free formaldehyde can be made by heating the solid PFA. 4% paraformaldehyde is usually made in PBS or TBS at 70 °C with several drops of 5N NaOH to help clarify the solution. Prepare 4% paraformaldehyde solution in a chemical hood and then store in a refrigerator. Because the solution will re-polymerize during storage it is best to use immediately or within a few days.

Additional resources

Before staining actin with phalloidin dye conjugates, how should I fixate my sample?