Fixation (histology)

In the fields of histology, pathology, and cell biology, fixation is a chemical process by which biological tissues are preserved from decay. Fixation terminates any ongoing biochemical reactions, and may also increase the mechanical strength or stability of the treated tissues.

Purpose of fixation
The purpose of fixation is to preserve a sample of biological material (tissue or cells) as close to its natural state as possible in the process of preparing tissue for examination. To achieve this goal, several conditions must usually be met.

First, a fixative usually acts to disable intrinsic biomolecules – particularly proteolytic enzymes – which would otherwise digest or damage the sample.

Second, a fixative will typically protect a sample from extrinsic damage. Fixatives are toxic to most common microorganisms (bacteria in particular) which might exist in a tissue sample or which might otherwise colonise the fixed tissue. In addition, many fixatives will chemically alter the fixed material to make it less palatable (either indigestible or toxic) to opportunistic microorganisms.

Finally, fixatives often alter the cells or tissues on a molecular level to increase their mechanical strength or stability. This increased strength and rigidity can help preserve the morphology (shape and structure) of the sample as it is processed for further analysis.

Even the most careful fixation does alter the sample and introduce artifacts that can interfere with interpretation of cellular ultrastructure. A prominent example is the bacterial "mesosome", which was thought to be an organelle in gram-positive bacteria in the 1970s, but was later shown by new techniques developed for electron microscopy to be simply an artifact of chemical fixation. Standardization of fixation and other tissue processing procedures takes this introduction of artifacts into account, by establishing what procedures introduce which kinds of artifacts. Researchers who know what types of artifacts to expect with each tissue type and processing technique can accurately interpret sections with artifacts, or choose techniques that minimize artifacts in areas of interest.

Fixation process
Fixation is usually the first stage in a multistep process to prepare a sample of biological material for microscopy or other analysis. Therefore, the choice of fixative and fixation protocol may depend on the additional processing steps and final analyses that are planned. For example, immunohistochemistry utilises antibodies which bind to a specific protein target. Prolonged fixation can chemically mask these targets and prevent antibody binding. In these cases, a 'quick fix' method using cold formalin for around 24 hours is typically used.

Types of fixation
There are generally three types of fixation process:

Perfusion: Fixation via bloodflow. The fixative is injected into the heart with the injection volume matching cardiac output. The fixative spreads through the entire body, and the tissue doesn't die until it is fixed. This has the advantage of preserving perfect morphology, but the disadvantages that the subject dies and the cost is high (because of the volume of fixative needed for larger organisms)

Immersion: The sample of tissue is immersed in fixative of volume at a minimum of 2/3rds greater than the volume of the tissue to be fixed. The fixative must diffuse through the tissue in order to fix, so tissue size and density, as well as the type of fixative must be taken into account. Using a larger sample means it will take longer for the fixative to reach the deeper tissue.

Crosslinking fixatives
Crosslinking fixatives act by creating covalent chemical bonds between proteins in tissue. This anchors soluble proteins to the cytoskeleton, and lends additional rigidity to the tissue.

Aldehydes
By far the most commonly used fixative in histology is the crosslinking fixative formaldehyde (often sold as a saturated aqueous solution under the name formalin). Formaldehyde is thought to interact primarily with the residues of the basic amino acid lysine.

Another popular aldehyde for fixation is glutaraldehyde. It is believed to operate by a similar mechanism to formaldehyde. As a somewhat larger molecule, glutaraldehyde may not penetrate thicker tissue specimens as effectively as formaldehyde. On the other hand, glutaraldehyde may offer a more rigid or tightly linked fixed product&mdash;its greater length and two aldehyde groups allow it to 'bridge' and link more distant pairs of protein molecules.

Some fixation protocols call for a combination of formaldehyde and glutaraldehyde, so that their respective strengths complement one another.

These crosslinking fixatives – especially formaldehyde – tend to preserve the secondary structure of proteins and may protect significant amounts of tertiary structure as well.

Oxidising agents
The oxidising fixatives can react with various side chains of proteins and other biomolecules, allowing the formation of crosslinks which stabilise tissue structure.

Osmium tetroxide is often used as a secondary fixative when samples are prepared for electron microscopy. (It is not used for light microscopy as it penetrates thick sections of tissue very poorly.)

Potassium dichromate, chromic acid, and potassium permanganate all find use in certain specific histological preparations.

Precipitating fixatives
Precipitating (or denaturing) fixatives act by reducing the solubility of protein molecules and (often) by disrupting the hydrophobic interactions which give many proteins their tertiary structure. The precipitation and aggregation of proteins is a very different process from the crosslinking which occurs with the aldehyde fixatives.

The most common precipitating fixatives are ethanol and methanol. Acetone is also used.

Acetic acid is a denaturant that is sometimes used in combination with the other precipitating fixatives. The alcohols, by themselves, are known to cause shrinkage of tissue during fixation while acetic acid alone is associated with tissue swelling; combining the two may result in better preservation of tissue morphology.

Other fixatives
Other fixative agents include picric acid and mercuric chloride.