The most common type of fixative used in histology is aldehydes. Aldehydes are compounds that have a carbon atom sharing a double bond with an oxygen atom, a single bond with a hydrogen atom, and a single bond with another atom or group of atoms. In the case of formaldehyde, that other atom is a hydrogen, CH2O. Formaldehyde is a gas that gets bubbled into the water with methanol to create the aqueous solution that we refer to as 100% formalin. 100% formalin is then diluted down with water to get the 10% formalin commonly used as a histology fixative. The percentage dilution of NBF (neutral buffered formalin) is based off of the type of tissue being fixed. Lower percentages of NBF will cause tissue swelling and higher percentages of NBF will cause tissue shrinkage. You would generally use a higher percentage of NBF such as 20% formalin when fixing something such as a whole brain, which has less water and more lipid content.
Alcoholic formalin is another aldehyde which is most often used as a secondary fixative after NBF to prevent primary alcoholic fixation. It will continue fixation and start the processing step. A major benefit of alcoholic formalin is that it will lower the freezing point, which makes it better for shipping in cold climates.
Although formaldehyde is the most common aldehyde you will see, it is not the only one. Glutaraldehyde and paraformaldehyde are additional aldehyde fixatives used most commonly in electron microscopy. Paraformaldehyde is similar to formaldehyde except that it does not contain methanol. You won’t see these used for routine histology because they can’t rapidly penetrate tissue. Glutaraldehyde’s larger size (it is a five carbon di-aldehyde) means that it penetrates and fixes slowly with less distortion to the tissue structure.
Mercuric chloride, a compound of mercury and chlorine can also be used to create fixatives. Mercuric chloride fixatives include Zenker’s fixative, which adds acetic acid, and Helly’s fixative, which adds formaldehyde. These fixatives are no longer widely used due to the toxicity of mercury when absorbed through the skin. Zinc sulfate has largely come to replace mercury as use of the zinc sulfate in fixatives gives great nuclear detail and no antigen retrieval is necessary in IHC. You may see zinc chloride used instead of sulfate but it is more corrosive and can cause damage to metal parts of tissue processors.
Picric acid is used to create several fixatives including Bouin’s solution, which contains picric acid, formaldehyde and acetic acid as well as Hollande’s fixative, which contains Copper acetate, formaldehyde and acetic acid. Acetic acid is commonly added to fixatives to counteract tissue shrinkage. Bouin’s is usually used in conjunction with the trichrome stain and is good for connective tissue. Hollande’s fixative is commonly used for gastric biopsies.
Zamboni is another solution that uses picric acid. It is made from paraformaldehyde and can be used for both electron and light microscopy.
There are a few things to be aware of when using picric acid. First, it can remove microcalcifications, which you may not want in some cases such as breast tissue. It is also explosive when dry, so appropriate safety considerations need to be taken.
As you may have surmised from the name, non-aqueous fixatives are those solutions that do not use water. Two of the most common of these are Carnoy’s and Clarke’s. Carnoy’s contains ethanol, chloroform, and acetic acid. Clarke’s is similar but does not use the chloroform. These are fast active fixatives, which makes them good for frozen sectioning. Check out our past post on Carnoy’s and Clarke’s solutions!
Want to learn more about types of fixatives? Check out NSH’s HT Prep Course, a course designed to prepare you for the HT ASCP exam.