We are all using some kind of fixative in the histology lab, but do we actually know what it’s doing to our cells and tissues? No matter what fixative we use, the purpose is to immobilize antigens and retain good cellular structure to allow us to do some kind of histology analysis.

Optomize your protocols

Usually we do fixation in order to do immunohistochemistry to allow us to investigate our tissue samples using antibodies. The whole process of fixation can be problematic as different epitopes require different fixation techniques and so this is yet another method that requires optimization.

Here is a brief guide to what happens to our samples in two of the main fixatives we use: PFA and Bouin’s solution.


Building bridges…

What exactly is PFA? PFA (or paraformaldehyde) is the condensation product of formaldehyde which is a water-soluble, colorless and toxic gas. Formaldehyde acts as a fixative because it reacts with the primary amines on proteins to form cross-links known as ‘methylene bridges’.

…to stabilize and preserve.

PFA adds to the side-chains of basic amino acids and to the amide nitrogen atoms of peptide linkages which stabilizes proteins and preserves morphology. Although it is a rapidly penetrating fixative, it cross-links proteins very slowly and often takes up to a week to achieve a good level of fixation. Consequently, one of the most common problems with PFA is under-fixation. Small pieces of tissue should ideally be immersed in PFA for at least 24h for a good level of preservation. Larger tissues, such as human biopsy samples, may need up to a few weeks in PFA to achieve adequate fixation.

Masking and hiding

Due to the formation of the protein cross-links during fixation, antigenic sites can be masked which can prevent recognition of the antigen by the antibody. This can be overcome by adding in an antigen retrieval step before immunohistochemistry protocols. This can be via a heat- or chemical-induced method. Both methods break the methylene bridges to expose antigenic sites to the antibodies.

Bouin’s Solution

Picric acid makes it yellow

Bouin’s fixative is a mixture of compounds and consists of formaldehyde, picric acid and acetic acid. Each component has a specific function that complements the other. The function of formaldehyde, which cross-links proteins, is discussed above. The role of the picric acid in this solution is to slowly penetrate into the tissue and cause coagulation of proteins by forming salts with basic proteins. This can, however, cause some shrinkage of the tissue. Picric acid is the component of Bouin’s solution that results in the characteristic yellow color.

Good for testis, not for blood!

Conversely, the acetic acid is rapidly penetrating and causes some swelling of the tissue, which partially counteracts the shrinkage caused by the picric acid. Acetic acid causes coagulation of nucleic acids and is often used where preservation of chromosomes is required. This makes it particularly useful in the fixation of the testis if visualization of the meiotic chromosomes is sought. If you are interested in red blood cells though, this is not the fixative for you; acetic acid lyses red blood cells and dissolves small iron and calcium deposits. In this case using some form of formaldehyde alone would be better.

Avoid autofluorescence

Bouin’s solution is a particularly good fixative for hematoxylin and eosin staining as it preserves the morphological detail very well. It is much better than PFA for fixing large tissues where the preservation of morphology is important, however, it is not the best choice for immunofluorescence due to the autofluorescence of picric acid.

Be careful with it

The standard fixation protocol for Bouin’s solution is perfusion followed by immersion, or immersion alone, for at least six hours. Large specimens can be kept in Bouin’s for longer, possibly up to three days, though it depends on the tissue. Due to the toxic nature of the components of Bouin’s solution, appropriate safety precautions should be taken. In particular, picric acid can be explosive and so proper disposal measures should be taken.

More fixatives, more choice

Fixation is not in any way restricted to these two solutions. There are many other fixatives we use every day such as methanol, acetone, glutaradleyde and many more. The choice depends on the starting cells or tissue and also the technique to be applied. As I mentioned above, some fixatives are better than other for fluorescence or histology analysis. As is usually the case in histology, trial and error play a big part in finding the best one for the job.