Fixation (histology)

In the fields of histology, pathology, and cell biology, fixation is a critical step in the preparation of histological sections by which biological tissues are preserved from decay, thereby preventing autolysis or putrefaction. The structure of a tissue is determined by the shapes and sizes of macromolecules in and around cells. The principal macromolecules inside a cell are proteins and nucleic acids. Fixation terminates any ongoing biochemical reactions, and may also increase the mechanical strength or stability of the treated tissues. The broad objective of tissue fixation is to preserve cells and tissue components and to do this in such a way as to allow for the preparation of thin, stained sections.

Purposes

In performing their protective role, fixatives denature proteins by coagulation, by forming additive compounds, or by a combination of coagulation and additive processes. A compound that adds chemically to macromolecules stabilizes structure most effectively if it is able to combine with parts of two different macromolecules, an effect known as cross-linking. Fixation of tissue is done for several reasons. One reason is to kill the tissue so that postmortem decay (autolysis and putrefaction) is prevented. Fixation preserves 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, several conditions usually must be met.

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

Second, a fixative typically protects a sample from extrinsic damage. Fixatives are toxic to most common microorganisms (bacteria in particular) that might exist in a tissue sample or which might otherwise colonize the fixed tissue. In addition, many fixatives 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.

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 uses antibodies that 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. Methanol (100%) can also be used for quick fixation, and that time can vary depending on the biological material. For example, MDA-MB 231 human breast cancer cells can be fixed for only 3 minutes with cold methanol (-20degrees Celsius). For enzyme localization studies, the tissues should either be pre-fixed lightly only, or post-fixed after the enzyme activity product has formed.

Types

There are generally three types of fixation process:

Heat fixation: After a smear has dried at room temperature, the slide is gripped by tongs or a clothespin and passed through the flame of a Bunsen burner several times to heat-kill and adhere the organism to the slide. Routinely used with bacteria and archaea. Heat fixation generally preserves overall morphology but not internal structures. Heat denatures the proteolytic enzyme and prevents autolysis. Heat fixation cannot be used in the capsular stain method as heat fixation will shrink or destroy the capsule (glycocalyx) and cannot be seen in stains.

Perfusion: Fixation via blood flow. 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 20 times greater than the volume of the tissue to be fixed. The fixative must diffuse through the tissue to fix, so tissue size and density, as well as type of fixative must be considered. Using a larger sample means it takes longer for the fixative to reach the deeper tissue. Best in a slight vacuum.

Chemical fixation

In this process, structures are preserved in a state (both chemically and structurally) as close to living tissue as possible. This requires a chemical fixative that can stabilise the proteins, nucleic acids and mucosubstances of the tissue by making them insoluble.

Types of chemical fixatives

Crosslinking fixatives - aldehydes

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.

By far the most commonly used fixative in histology is formaldehyde. It is usually used as a 10% Neutral Buffered Formalin (NBF), that is approx. 3.7%-4.0% formaldehyde in phosphate buffered saline. Because formaldehyde is a gas at room temperature, formalin-formaldehyde gas dissolved in water (~37% w/v)-is used when making the former fixative. Paraformaldehyde is a polymerized form of formaldehyde, usually obtained as a fine white powder, which depolymerises back to formalin when heated. Formaldehyde fixes tissue by cross-linking the proteins, primarily the residues of the basic amino acid lysine. Its effects are reversible by excess water and it avoids formalin pigmentation. Other benefits include: Long term storage and good tissue penetration. It is particularly good for immunohistochemistry techniques. Also the formaldehyde vapor can be used as a fixative for cell smears.

Another popular aldehyde for fixation is glutaraldehyde. It operates in a similar way to formaldehyde by causing deformation of the alpha-helix structures in proteins. However glutaraldehyde is a larger molecule, and so its rate of diffusion across membranes is slower than formaldehyde. Consequently glutaraldehyde fixation on thicker tissue samples may be hampered, but this problem can be overcome by reducing the size of the tissue sample. One of the advantages of glutaraldehyde fixation is that it may offer a more rigid or tightly linked fixed product—its greater length and two aldehyde groups allow it to 'bridge' and link more distant pairs of protein molecules. It causes rapid and irreversible changes, fixes quickly, is well suited for electron microscopy, fixes well at 4^oC, and gives best overall cytoplasmic and nuclear detail. However it is not ideal for immunohistochemistry staining.

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.

Precipitating fixatives - alcohols

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

The most common precipitating fixatives are ethanol and methanol. They are commonly used to fix frozen sections and smears. Acetone is also used and has been shown to produce better histological preservation than frozen sections when employed in the Acetone Methylbenzoate Xylene (AMEX) technique.

The protein denaturants - methanol, ethanol and acetone - are rarely used alone for fixing blocks unless studying nucleic acids.

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

Oxidizing agents

The oxidizing fixatives can react with various side chains of proteins and other biomolecules, allowing formation of crosslinks that stabilize tissue structure. However they cause extensive denaturation despite preserving fine cell structure and are used mainly as secondary fixatives.

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.

Mercurials

Mercurials such as B-5 and Zenker's fixative have an unknown mechanism that increases staining brightness and give excellent nuclear detail. Despite being fast, mercurials penetrate poorly and produce tissue shrinkage. Their best application is for fixation of hematopoietic and reticuloendothelial tissues. Also note that since they contain mercury care must be taken with disposal.

Picrates

Picrates penetrate tissue well to react with histones and basic proteins to form crystalline picrates with amino acids and precipitate all proteins. It is a good fixative for connective tissue, preserves glycogen well, and extracts lipids to give superior results to formaldehyde in immunostaining of biogenic and polypeptide hormones However, it causes a loss of basophilia unless the specimen is thoroughly washed following fixation.

HOPE fixative

Hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE) gives formalin-like morphology, excellent preservation of protein antigens for immunohistochemistry and enzyme histochemistry, good RNA and DNA yields and absence of crosslinking proteins.

Frozen sections

Small pieces of tissue (5×5×3mm) are placed in a cryoprotective embedding medium—OCT, TBS, or Cryogel—then snap frozen in isopentane cooled by liquid nitrogen. Tissue is then sectioned in a freezing microtome or cryostat. Sections are then fixed in one of the following fixatives: Absolute acetone for 10–15 seconds, 95% ethanol for 10–15 seconds or Absolute acetone 10 seconds followed by 95% ethanol 10 seconds

Advantages

• Give better preservation of antigenicity
• Minimal exposure to fixative
• Not exposed to the organic solvents
• Much faster than other forms of fixations.

Disadvantages

• Lack morphological detail
• Presents a potential Biohazard

Target and chemical fixative do's and don'ts

Target	Fixative of choice	Fixative to avoid
Proteins	Neutral buffered formalin, Paraformaldehyde	Osmium tetroxide
Enzymes	Frozen sections	Chemical fixatives
Lipids	Frozen sections*, glutaraldehyde/osmium tetroxide	Alcoholic fixatives, neutral buffered formalin
Nucleic acids	Alcoholic fixatives, HOPE	Aldehyde fixatives
Mucopolysaccharides	Frozen sections	Chemical fixatives
Biogenic amines	Bouin solution~, Neutral buffered formalin
Glycogen	Alcoholic based fixatives	Osmium tetroxide

• Frozen sections preserve RNA and Lipids despite poor morphology. Compare to paraffin sections, synonymous to chemical fixatives in the table, which destroy RNA and affect some antigens BUT give good morphology.

~ A picrate

Factors affecting fixation

pH

Should be kept in the physiological range, between pH 4-9. The pH for the ultrastructure preservation should be buffered between 7.2 to 7.4

Osmolarity

Hypertonic solutions give rise to cell shrinkage.

Hypotonic solutions result in cell swelling and poor fixation.

10% neutral buffer formalin is 4% formaldehyde (1.33 osmolar) in PBS buffer (0.3 osmolar) sums to 1.63 osmolar. This is a very hypertonic solution yet it has worked well as a general tissue fixation condition for many years in pathology labs. The size of tissue can also effect the fixation process.

Size of the specimen

1–4 mm thickness [0.5 cm]

Volume of the fixative

At least 15-20 times greater than tissue volume

Temperature

Increasing the temperature increases speed of fixation. However, care is required to avoid cooking the specimen. Fixation is routinely carried out at room temperature.

Duration

As a general rule, 1hr per 1mm of tissue PFA must penetrate. So if we have a three-dimensional tissue block, it is the shortest dimension that determines fixation time.

Time from removal to fixation

Fixation is a chemical process, and time must be allowed for the process to complete. Although "over fixation" can be detrimental, under-fixation has recently been appreciated as a significant problem and may be responsible for inappropriate results for some assays.

External links

• Fixing specimens for making permanent slides
• Fixation strategies and formulations for immunohistochemical staining