Fixation (histology)
Encyclopedia
In the fields of histology
, pathology
, and cell biology
, fixation is a chemical process by which biological tissue
s are preserved from decay, thereby preventing autolysis
or putrefaction
. Fixation terminates any ongoing biochemical reactions, and may also increase the mechanical strength or stability of the treated tissues.
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
enzyme
s—which otherwise digests or damages 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 colonise 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.
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.
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 prevent 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 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 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.
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 aprox. 3.7% 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 polymerised 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 vapour can be used as a fixatives 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 4oC, 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 protein
s and may protect significant amounts of tertiary structure
as well.
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. 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
.
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.
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 minutes,
95% ethanol for 10–15 minutes or
Absolute acetone 10 minutes followed by 95% ethanol 10 minutes
~ A picrate
Hypotonic solutions result in cell swelling and poor fixation.
Histology
Histology is the study of the microscopic anatomy of cells and tissues of plants and animals. It is performed by examining cells and tissues commonly by sectioning and staining; followed by examination under a light microscope or electron microscope...
, pathology
Pathology
Pathology is the precise study and diagnosis of disease. The word pathology is from Ancient Greek , pathos, "feeling, suffering"; and , -logia, "the study of". Pathologization, to pathologize, refers to the process of defining a condition or behavior as pathological, e.g. pathological gambling....
, and cell biology
Cell biology
Cell biology is a scientific discipline that studies cells – their physiological properties, their structure, the organelles they contain, interactions with their environment, their life cycle, division and death. This is done both on a microscopic and molecular level...
, fixation is a chemical process by which biological tissue
Biological tissue
Tissue is a cellular organizational level intermediate between cells and a complete organism. A tissue is an ensemble of cells, not necessarily identical, but from the same origin, that together carry out a specific function. These are called tissues because of their identical functioning...
s are preserved from decay, thereby preventing autolysis
Autolysis (biology)
In biology, autolysis, more commonly known as self-digestion, refers to the destruction of a cell through the action of its own enzymes. It may also refer to the digestion of an enzyme by another molecule of the same enzyme....
or putrefaction
Putrefaction
Putrefaction is one of seven stages in the decomposition of the body of a dead animal. It can be viewed, in broad terms, as the decomposition of proteins, in a process that results in the eventual breakdown of cohesion between tissues and the liquefaction of most organs.-Description:In terms of...
. Fixation terminates any ongoing biochemical reactions, and may also increase the mechanical strength or stability of the treated tissues.
Purposes of fixation
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
Biological tissue
Tissue is a cellular organizational level intermediate between cells and a complete organism. A tissue is an ensemble of cells, not necessarily identical, but from the same origin, that together carry out a specific function. These are called tissues because of their identical functioning...
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
Proteolysis
Proteolysis is the directed degradation of proteins by cellular enzymes called proteases or by intramolecular digestion.-Purposes:Proteolysis is used by the cell for several purposes...
enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
s—which otherwise digests or damages the sample.
Second, a fixative typically protects a sample from extrinsic damage. Fixatives are toxic to most common microorganisms (bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
in particular) that might exist in a tissue sample or which might otherwise colonise 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
Morphology (biology)
In biology, morphology is a branch of bioscience dealing with the study of the form and structure of organisms and their specific structural features....
(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
Mesosome
Mesosomes are folded invaginations in the plasma membrane of bacteria that are produced by the chemical fixation techniques used to prepare samples for electron microscopy...
, which was thought to be an organelle
Organelle
In cell biology, an organelle is a specialized subunit within a cell that has a specific function, and is usually separately enclosed within its own lipid bilayer....
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 microscopyMicroscopy
Microscopy is the technical field of using microscopes to view samples and objects that cannot be seen with the unaided eye...
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.
Types of fixation
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 prevent autolysis. Heat fixation cannot be used in the capsular stain method as heat fixation will shrink or destroy the capsule (glycocalyx
Glycocalyx
Glycocalyx is a general term referring to extracellular polymeric material produced by some bacteria, epithelia and other cells. The slime on the outside of a fish is considered a glycocalyx. The term was initially applied to the polysaccharide matrix excreted by epithelial cells forming a...
) and cannot be seen in stains.
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 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.Crosslinking fixatives - Aldehydes
Crosslinking fixatives act by creating covalent chemical bondsCovalent bond
A covalent bond is a form of chemical bonding that is characterized by the sharing of pairs of electrons between atoms. The stable balance of attractive and repulsive forces between atoms when they share electrons is known as covalent bonding....
between proteins in tissue. This anchors soluble proteins to the cytoskeleton
Cytoskeleton
The cytoskeleton is a cellular "scaffolding" or "skeleton" contained within a cell's cytoplasm and is made out of protein. The cytoskeleton is present in all cells; it was once thought to be unique to eukaryotes, but recent research has identified the prokaryotic cytoskeleton...
, and lends additional rigidity to the tissue.
By far the most commonly used fixative in histology is formaldehyde
Formaldehyde
Formaldehyde is an organic compound with the formula CH2O. It is the simplest aldehyde, hence its systematic name methanal.Formaldehyde is a colorless gas with a characteristic pungent odor. It is an important precursor to many other chemical compounds, especially for polymers...
. It is usually used as a 10% Neutral Buffered Formalin (NBF), that is aprox. 3.7% formaldehyde in phosphate buffered saline
Phosphate buffered saline
Phosphate buffered saline is a buffer solution commonly used in biological research. It is a water-based salt solution containing sodium chloride, sodium phosphate, and, in some formulations, potassium chloride and potassium phosphate. The buffer's phosphate groups help to maintain a constant pH...
. 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 polymerised 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
Lysine
Lysine is an α-amino acid with the chemical formula HO2CCH4NH2. It is an essential amino acid, which means that the human body cannot synthesize it. Its codons are AAA and AAG....
. 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 vapour can be used as a fixatives for cell smears.
Another popular aldehyde
Aldehyde
An aldehyde is an organic compound containing a formyl group. This functional group, with the structure R-CHO, consists of a carbonyl center bonded to hydrogen and an R group....
for fixation is glutaraldehyde
Glutaraldehyde
Glutaraldehyde is an organic compound with the formula CH22. A pungent colorless oily liquid, glutaraldehyde is used to disinfect medical and dental equipment...
. 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 4oC, 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
Secondary structure
In biochemistry and structural biology, secondary structure is the general three-dimensional form of local segments of biopolymers such as proteins and nucleic acids...
of protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s and may protect significant amounts of tertiary structure
Tertiary structure
In biochemistry and molecular biology, the tertiary structure of a protein or any other macromolecule is its three-dimensional structure, as defined by the atomic coordinates.-Relationship to primary 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 precipitationPrecipitation (chemistry)
Precipitation is the formation of a solid in a solution or inside anothersolid during a chemical reaction or by diffusion in a solid. When the reaction occurs in a liquid, the solid formed is called the precipitate, or when compacted by a centrifuge, a pellet. The liquid remaining above the solid...
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
Ethanol
Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a...
and methanol
Methanol
Methanol, also known as methyl alcohol, wood alcohol, wood naphtha or wood spirits, is a chemical with the formula CH3OH . It is the simplest alcohol, and is a light, volatile, colorless, flammable liquid with a distinctive odor very similar to, but slightly sweeter than, ethanol...
. They are commonly used to fix frozen sections and smears. Acetone
Acetone
Acetone is the organic compound with the formula 2CO, a colorless, mobile, flammable liquid, the simplest example of the ketones.Acetone is miscible with water and serves as an important solvent in its own right, typically as the solvent of choice for cleaning purposes in the laboratory...
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
Acetic acid
Acetic acid is an organic compound with the chemical formula CH3CO2H . It is a colourless liquid that when undiluted is also called glacial acetic acid. Acetic acid is the main component of vinegar , and has a distinctive sour taste and pungent smell...
is a denaturant that is sometimes used in combination with the other precipitating fixatives. 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
Morphology (biology)
In biology, morphology is a branch of bioscience dealing with the study of the form and structure of organisms and their specific structural features....
.
Oxidising agents
The oxidising 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
Chromic acid
The term chromic acid is usually used for a mixture made by adding concentrated sulfuric acid to a dichromate, which may contain a variety of compounds, including solid chromium trioxide. This kind of chromic acid may be used as a cleaning mixture for glass. Chromic acid may also refer to the...
, and potassium permanganate
Potassium permanganate
Potassium permanganate is an inorganic chemical compound with the formula KMnO4. It is a salt consisting of K+ and MnO4− ions. Formerly known as permanganate of potash or Condy's crystals, it is a strong oxidizing agent. It dissolves in water to give intensely purple solutions, the...
all find use in certain specific histological preparations.
Mercurials
Mercurials such as B-5 and Zenker's 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 isopentaneIsopentane
Isopentane, C5H12, also called methylbutane or 2-methylbutane, is a branched-chain alkane with five carbon atoms. Isopentane is an extremely volatile and extremely flammable liquid at room temperature and pressure. The normal boiling point is just a few degrees above room temperature and...
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 minutes,
95% ethanol for 10–15 minutes or
Absolute acetone 10 minutes followed by 95% ethanol 10 minutes
Advantages
- Give better preservation of antigenicity
- Minimal exposure to fixative
- Not exposed to the organic solvents
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 Bouin Solution Bouin solution is a compound fixative used in histology. It is composed of picric acid, acetic acid and formaldehyde in an aqueous solution. It is especially good for gastrointestinal tract biopsies because this fixative allows crisper and better nuclear staining than 10% neutral-buffered formalin... ~, 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
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 7.4Osmolarity
Hypertonic solutions give rise to cell shrinkage.Hypotonic solutions result in cell swelling and poor fixation.