Fixation Strategies and Formulations - US (2024)

For immunohistochemistry (IHC) to succeed, it is essential that the morphology of the tissues and cells is retained and that the antigenic sites remain accessible to the detection reagents being used.

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Page contents

• Introduction
• Chemical vs physical fixation
• Formaldehyde, glutaraldehyde, and other chemical fixatives
• Fixative formulations for specific applications

Fixation plays four critical roles in immunohistochemistry:

• Itpreserves andstabilizes cell morphology and tissue architecture
• Itinactivatesproteolytic enzymes that could otherwise degrade the sample
• Itstrengthens samplesso that they canwithstand further processing and staining
• Itprotects samples against microbial contamination andpossibledecomposition.

The right fixation method requires optimization based on the application and the target antigen to be stained.This means that the optimal fixation method may have to be determined empirically.Common methods of fixation include:

• Perfusion: Tissues can be perfused with fixative following exsanguination and saline perfusion to allow rapid fixation of entire organs.
• Immersion: Samples are immersed in fixative which then diffuses into and through the tissue or cell sample. Immersion is often combined with perfusion to ensure thorough fixation throughout the tissue.
• Freezing: Samples with antigens that are toolabilefor chemical fixation or exposure to the organic solvents used for de-paraffinization can be embedded inacryoprotective embedding medium, such asoptimal cutting temperature (OCT)compound, and then snap-frozen and stored in liquid nitrogen.
• Drying: Blood smears for ICCstainingare air-dried and waved across a flame to heat-fix the cells to the slide.

While a particular fixative may preserve the immunoreactivity of oneantigenicepitope, it may destroy others, even if they are on the same antigen. The guidelinesprovided hereare helpful in determining the appropriate fixative for a particular system, butit is important to remember thateach antigen is unique. Therefore, the following considerations should be addressed when choosing a fixative:

• Type of fixative (formaldehyde, glutaraldehyde,organic solvent,etc.)
• Rate of penetration and fixation
• Fixative concentration
• FixativepH
• Ideal FixationTemperature
• Post-fixation treatment

Chemical vs physical fixation

Chemical fixativescrosslink or precipitate sample proteins, which can mask target antigens or prevent antibody accessibility to the tissue target after prolonged fixation. No single fixative is ideal for all tissues, samples or antigens.This means that each fixationproceduremust beoptimized toassureadequatefixationwithoutalteringtheantigen or disturbing the endogenous location and the cellular detail of the tissue.

Physical fixation is an alternate approach to prepare samples for staining, and the specific method depends on the sample source and the stability of the target antigen. For example, blood smears areusuallyfixed by drying, which removes the liquidfromthesample and fixesthe cellsto the slide. Tissues that are too delicate for the rigorous processing involved with paraffin removal and antigen retrieval are first embedded in cryoprotective embedding medium, such as OCTcompound, and then snap-frozen and stored in liquid nitrogen untilthey aresectioned.The example below provides an example of IHC staining in formalin fixed tissue.

IHC was performed onaformalin fixed, paraffin embedded (FFPE)human colon cancer tissue section.To expose target proteins, heat-induced epitope retrieval(HIER)was performed using 10mM sodium citratebuffer, pH 6,(e.g., 00-5000, AP-9003-125)for 20 minby heatingat 95°C. Following antigen retrieval,cooling to roomtemperatureand washing,tissues were blocked in 3% BSA (Product # 37525) in PBST for 30 min at room temperature and then probed with an Ezrin monoclonal antibody (Product # MA5-13862) at a dilution of 1:100 for 1 h in a humidified chamber. Tissues were washed extensively with PBS/0.025% Tween-20 (Product #003005) and endogenous peroxidase activitywasquenched with Peroxidase Suppressor (Product # 35000) for 30 min at room temperature. Detection was performed using an HRP-conjugated goat anti-mouse IgG-HRP secondary antibody (Product # 31430) at a dilution of 1:500 followed by colorimetric detection using Metal Enhanced DAB Substrate Kit (Product # 34065). Images were taken on a lightmicroscope at 40X magnification.

Formaldehyde, glutaraldehyde and other chemical fixatives

Formaldehyde

The most widely used chemical fixative is formaldehyde, which shows broad specificity for most cellular targets. The water-soluble, colorless, toxic, and pungent gas reacts with primary amines on proteins and nucleic acids to form partially-reversible methylene bridgecrosslinks

Formaldehyde andparaformaldehyde

Most commercial formaldehyde is preparedfromparaformaldehyde (PFA,polymeric formaldehyde) dissolved in distilled/deionized water, withup to 10% (v/v)methanol added to stabilize the aqueousformaldehyde.Stabilizationis important to prevent oxidationof the formaldehydeto formic acid anditseventual re-polymerization toparaformaldehyde.Toavoid using methanol-stabilizedformaldehyde for fixation, many protocols recommend making “fresh” formaldehyde from paraformaldehyde immediately before sample fixation.

Formalin vs.formaldehyde

The terms “formalin” and “formaldehyde” are often used interchangeably, although the chemical composition of each fixative is different.Formalin is made with formaldehyde but the percentage denotes a different formaldehyde concentration thantrueformaldehyde solutions.For example, 10% neutral-buffered formalin (NBF,or simply formalin) is really a 4%(v/v)formaldehyde solution.The basis for this difference is that historically, formalin was prepared with commercial-grade stock formaldehyde, whichwas 37 to 40%(w/v)formaldehyde, by diluting it 1:10withphosphatebufferat neutral pH

Glutaraldehyde

Glutaraldehyde is a dialdehydecompoundthat reacts with aminoandsulfhydrylgroups and possibly with aromatic ring structures. Fixatives containing glutaraldehyde are stronger protein crosslinkers than formaldehyde.However, they penetrate tissue more slowly, causing extraction of soluble antigens and modification of thetissue architecture. Tissues that have been fixed with a glutaraldehyde-based fixative must be treatedor quenchedwith inert amine-containing molecules prior to theIHCstainingbecauseanyfree, unsaturated aldehyde groupsthatare availablewill reactcovalently withamine-containing moieties such as antibodies(Schiff base formation). The most efficient aldehyde blockers/quenchersare ethanolamine and lysine.

Otherfixatives

Mercuric chloride-based fixatives aresometimesused as alternativesto aldehyde-based fixatives to overcome poor cytological preservation. Theseharshfixatives work byreacting with amines, amides, amino acids like cysteine, and phosphate groups in proteins and nucleic acids. The result is protein and nucleicacidcoagulation, which can lead toundesirabletissuehardening.Thebenefitsofusing these fixativesaremore intenseIHCstainingaccompanied bythe preservation of cytological detail allowing for easier morphological interpretation. These fixatives oftenincludeneutralsaltscontaining zincto maintain tonicity andtheycan be mixed with other fixatives to provide a balanced, lessharsh formulation.Mercuric chloride-based fixatives include Helly and Zenker's Solution.One disadvantage of mercury-containing fixatives is that sections must be cleared of mercury deposits beforeIHC staining. The main disadvantageof these mercurybased fixativesis that theyare highly toxic, corrosive, andtheyrequire special disposal procedures. For this reason, they are not used frequently any more.

Precipitating fixatives include ethanol, methanol and acetone. Thesesolventsprecipitateand coagulatelarge protein molecules, thereby denaturingthem,andcan begood for cytological preservation.Suchreagents can also permeabilize cells, which may be critical depending on the sample.However, acetone, in particular,extractslipids from cells and tissues, which can adversely affect morphology.Despite this fact, acetone is usually used as a post-fixative for frozen sections that have already been bound to slides. In contrast, the solvent fixativesare notappropriatefor electronmicroscopy becausetheycancauseseveretissue shrinkage.

Diimidoesterfixation using dimethylsuberimidate(DMS), an amine-reactive crosslinker, is ararely-usedalternative to aldehyde-based fixation (Hassel, J. et al., 1974). DMS is ahom*obifunctionalreagentwhichcrosslinkstheαand ε-amino groups of proteinsto each other.Diimidoestersare unique in that they create amidine linkages withtheamineson thetargetmolecules. As a result, DMS does notchangethe net charge of the protein. The advantages of using DMS as a fixative for both light and electron microscopy include retention of immunoreactivity of the antigen and the lack of aldehyde groups that require blocking.

There are a variety of other fixatives that are used in special situations. These includeacrolein and glyoxal, which are similar to formaldehyde, andosmium tetroxide, which is particularly well-suited as a fixative prior to electron microscopy. Otherspecialtyfixativeincludecarbodiimide and other protein crosslinkers, zinc salt solutions, picric acid, potassium dichromate, and acetic acid.

Fixative formulations for specific applications

While histochemistry and histopathology texts describe many different fixatives and their effects on various tissue components, the most common fixatives and their general target antigens are listed below. Formulations for common fixatives then follow.

Fixatives commonly used for particular kinds of antigens

Recommended reading

1. Hassel, J. and Hand, A.R. (1974). J. Histochem. Cytochem. 22 229-239.
2. Coons, A.A.,et al. (1942)J. Immunol.45, 159-170
3. Beisker W et al. (1987) Cytometry8:235–239.
4. Cowen Tet al. (1985)Histochemistry82:205–208.
5. Mosiman VLet al. (1997)Cytometry30:151–156.
6. Romijn, Herms J.et al. (1999)J Histochem Cytochem47:229–236.