Whenever you work in the histology lab, there is a great chance that you’ll be using some type of fixatives before you can proceed with your experiment. By definition, fixation is the process of preserving biological tissues by terminating any biochemical reactions thereby preventing autolysis and putrefaction. It also preserves the integrity and morphology of the sample by inhibiting bacterial and fungal growth.
By successfully killing the biological material (e.g., tissue) before postmortem decay can take place, you can preserve its natural state while increasing its mechanical strength. This is extremely important since you’ll need an extremely stable material if you want to preserve the shape and structure of your sample.
General Types of Fixation
Since fixation is usually just the first of many steps involved in preparing a biological material for analysis, your choice of fixative and fixation process ultimately depends on what you want to accomplish. Depending on your specimen, you can choose one of the three general types of fixation processes – heat fixation, perfusion fixation, and immersion fixation.
In heat fixation, the slide containing the sample is heated by passing it through the flames of a Bunsen burner several times to denature the proteolytic enzyme and prevent autolysis. This process can adequately preserve the morphology of the sample (but not the internal structures) and is usually used for preparing bacterial and archaea samples.
Perfusion fixation can best be described as fixation through blood flow. The fixative is injected into the heart and spreads through the entire body. Since the tissue doesn’t die until it is fixed, you can get a sample with perfect morphology. Unfortunately, the subject dies during the process.
Immersion fixation involves the use of fixative solutions. Since the solution should completely diffuse through the sample, the size and density of the sample should be a major consideration. For best results, the sample should be immersed in the appropriate fixative at a minimum volume of 20 times greater than the sample.
Popular Fixative Solutions
Aldehydes work by creating covalent bonds between proteins (particularly between lysine residues) in the tissue sample which increases the rigidity of the structure.
- Formaldehyde (10% neutral buffered formalin) is by far the most popular fixative used in histology since it penetrates the tissue well and creates crosslinks without affecting the sample tissue’s antigenicity. While it is relatively slow to fix, it is highly recommended for immunohistochemical techniques.
- Glutaraldehyde fixes quickly and provides great cytoplasmic and nuclear detail, but it penetrates poorly and deforms the alpha helix structure in proteins. As such, it is good for electron microscopy but not so for immunohistochemical staining.
- Paraformaldehyde (PFA) is an effective fixative that reacts with primary amines found in the protein to form crosslinks (“methylene bridges”). PFA works great in stabilizing proteins and preserving morphology, but it fixes very slowly (more than 24 hours for smaller tissues and up to a few weeks for larger tissues) and may mask antigenic sites.
Picrates satisfactorily penetrate tissue samples and are ideal fixatives for connective tissues. A good example of picrates is Bouin’s solution (a mixture composed of 40% formaldehyde, picric acid saturated aqueous solution, glacial acetic acid). Formaldehyde works by forming crosslinks between proteins in the sample while picric acid slowly penetrates the tissues to coagulate the proteins and form salts with basic proteins. Acetic acid counters the shrinkage caused by picric acid and coagulates nucleic acids (useful for visualizing meiotic chromosomes). However, it is not recommended when working with red blood cells. Bouin’s solution is explosive so take extra precaution when using it.
Alcohols act by reducing the solubility and disrupting hydrophobic interactions between protein molecules. Ethanol and methanol, the most popular fixatives in this group, are commonly used for fixing cytologic smears and frozen sections since they act quickly and provide excellent nuclear detail. However, they are rarely used alone since they may cause tissue shrinkage and hardening.
Oxidizing agents such as osmium tetroxide, potassium dichromate, potassium permanganate, and chromic acid allow for the formation of stabilizing crosslinks but may cause extensive denaturation. As such, they are primarily used as secondary fixatives.
While the mechanism by which mercurials such as Zenker’s fixative and B-5 fixes tissues is still largely unknown up to now, these fixatives penetrate fast and produce excellent nuclear detail. However, they penetrate rather poorly and cause tissue shrinkage. They also contain mercury, so extra care is needed when using them.
