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

Introduction

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

Learn more

  • IHCTroubleshootingGuide

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.

Fixation Strategies and Formulations - US (1)

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

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  • Application Note: Enhanced Immunohistochemistry

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.

Learn more

  • Immunohistochemistry Troubleshooting Guide

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.

Sample Type or Antigen

Fixative

Most proteins, peptides and enzymes of low molecular weight

4%(w/v)Paraformaldehyde 
4%(w/v)Paraformaldehyde-1%(v/v)glutaraldehyde
10% Neutral-bufferedformalin (NBF)

Delicate tissue

Bouin'sfixative

Small molecules such as amino acids

4%(w/v)Paraformaldehyde-1%(v/v) glutaraldehyde

Blood-forming organs (e.g.liver, spleen, bone marrow); connective tissue

Zenker'ssolution
Hellysolution

Nucleic acids

Carnoy'ssolution

Large protein antigens (e.g.,immunoglobulins)

Ice-coldacetone(100%)ormethanol (100%)

Ideal for electron microscopy4%(w/v)Paraformaldehyde-1%(v/v)glutaraldehyde; 1% (w/v) osmium tetroxide

Fixatives commonly used for particular kinds of antigens

4% (w/v) Paraformaldehyde in 0.1 M phosphate buffer
Mix together:
  • NaH2PO4, 3.2 g
  • Na2HPO4, 10.9 g
  • Distilled water, 1000 mL
Adjust pH to 7.4. Then add:
  • Paraformaldehyde, 40 g

Heat mixture to 60°C while stirring and add 1-2 drops of 1 N NaOH to help the paraformaldehyde to dissolve. Cool and filter the solution.

4% Paraformaldehyde-1% glutaraldehyde in 0.1 M phosphate buffer
Prepare 4% paraformaldehyde in 0.1 M phosphate buffer, as above. Then add:
  • Glutaraldehyde, 20 mL
Bouin's fixative
Mix together:
  • Saturated aqueous picric acid, 750 mL
  • 40% (w/v) formaldehyde, 250 mL
  • Glacial acetic acid, 50 mL

Store at room temperature

10% Neutral-buffered formalin
Mix together:
  • Na2HPO4, anhydrous, 6.5 g
  • NaH2PO4•H20, 4 g
  • Distilled water, 900 mL
Adjust pH to 7.4, Then add:
  • 40% (w/v) formaldehyde, 100 mL

Store at 4°C

Zenker's solution
Mix together:
  • Mercuric chloride, 5 g
  • Potassium dichromate, 2.5 g
  • Sodium sulfate decahydrate, 1 g
  • Distilled water, 100 mL
  • Acetic acid, glacial, 5 mL*

Mix thoroughly to dissolve components. Wash sample for 24 h with distilled water after fixation. Never use metal forceps to handle tissue because they will corrode.

Precipitating solutions
Prepare: Ice-cold acetone or methanol (100%)Fix for 5-10 min at room temperature. Great for fixing and permeabilizing, if needed.
*Add component right before use

Common fixative formulations and notes on their storage and use

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.
Fixation Strategies and Formulations - US (2024)

FAQs

What are different fixation techniques? ›

The fixation methods are classified into chemical fixation and physical fixation. The former method chemically fixes proteins, lipids, etc., by using chemicals and the latter method physically fixes water in cells or tissues by freezing them.

What is the 4 PFA fixation protocol for flow cytometry? ›

4% PFA fixation protocol

1.1 Wash cells in x1 PBS and pellet cells by centrifugation (typically, ~2-5 mins at 200-300g is sufficient). Discard supernatant. 1.2 Add 4% PFA (ice cold), ~100 µl per tube, gently vortex to ensure pellet is separated. Incubate for 20 mins at room temperature.

What are the methods of IHC fixation? ›

Fixing Samples for IHC

For the best results, tissue should undergo rapid and uniform fixation. This can be achieved by two methods: whole animal perfusion or tissue/organ immersion in fixative.

What are the methods of cell fixation? ›

Types of fixation

Fixation of tissues can be achieved by chemical or physical means. Physical methods include heating, micro-waving and cryo-preservation (freeze drying). Heat fixation is rarely used on tissue specimens, its application being confined to smears of micro organisms.

What are the basic concepts of fixation? ›

FIXATION. Fixation of tissues is the most crucial step in the preparation of tissue for observation in the transmission electron microscope. Fixation consists of two steps: cessation of normal life functions in the tissue (killing) and stabilization of the structure of the tissue (preservation).

What are the principles of fixation technique? ›

5.3 PRINCIPLE OF FIXATION

Fixation results in denaturation and coagulation of protein in the tissues. The fixatives have a property of forming cross links between proteins, thereby forming a gel, keeping everything in their in vivo relation to each other.

What is the most common fixative used in immunohistochemistry? ›

10% neutral buffered formalin (NBF) is most commonly used. Where our datasheets state IHC-P as a tested application, this fixative has been used unless stated otherwise. Other fixatives such as Bouin solution (paraformaldehyde/picric acid) are used less frequently.

What are the methods of fixation for immunofluorescence? ›

Fixation. The cells may be fixed using one of two methods: Incubating the cells in 100% methanol (chilled at -20°C) at room temperature for 5 min. Using 4% paraformaldehyde in PBS pH 7.4 for 10 min at room temperature.

What is the best histological fixation method for tissue? ›

Fix tissue in 10% neutral buffer formaldehyde (NBF) solution or freshly prepared 4% paraformaldehyde solution. Formaldehyde-based fixatives are preferred for long term tissue preservation and are known to produce best results for sectioning, morphology (H&E), special stains and immunohistochemistry.

What are the types of chemical fixation? ›

Chemical fixation
  • Crosslinking fixatives – aldehydes.
  • Precipitating fixatives – alcohols.
  • Oxidizing agents.
  • Mercurials.
  • Picrates.
  • HOPE fixative.

What are simple fixatives? ›

Simple fixative is a solution or gas which contains only one active ingredient or that has a singlechemical solution. Examples include; Formaldehyde, Glutaraldehyde, Mercuric Chloride, Potassium Dichromate, Picric Acid, Osmium Tetroxide, Acetic Acid, Ethanol, Acetone and Chromic Acid.

What is the best fixative for cells? ›

Aldehydes, such as formalin or formaldehyde, are the preferred fixative for preserving cell morphology and are well suited for immunostaining of membrane proteins.

What are the methods of brain fixation? ›

Brains are fixed by perfusion or by immersion. Fixation by perfusion yields more uniform fixation than immersion (McFadden et al.

What are the different types of smear fixation? ›

Fix the smear to the slide using heat fixation or methanol fixation according to your laboratory's procedure.

What are the different types of bacterial fixation? ›

Two kinds of nitrogen-fixing microorganisms are recognized: free-living (nonsymbiotic) bacteria, including the cyanobacteria (or blue-green algae) Anabaena and Nostoc and genera such as Azotobacter, Beijerinckia, and Clostridium; and mutualistic (symbiotic) bacteria such as Rhizobium, associated with leguminous plants, ...

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