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.

Introduction

Fixation plays four critical roles in immunohistochemistry:

  • It preserves and stabilizes cell morphology and tissue architecture
  • It inactivates proteolytic enzymes that could otherwise degrade the sample
  • It strengthens samples so that they can withstand further processing and staining
  • It protects samples against microbial contamination and possible decomposition. 

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 too labile for chemical fixation or exposure to the organic solvents used for de-paraffinization can be embedded in a cryoprotective embedding medium, such as optimal cutting temperature (OCT) compound, and then snap-frozen and stored in liquid nitrogen.
  • Drying: Blood smears for ICC staining are air-dried and waved across a flame to heat-fix the cells to the slide.

While a particular fixative may preserve the immunoreactivity of one antigenic epitope, it may destroy others, even if they are on the same antigen. The guidelines provided here are helpful in determining the appropriate fixative for a particular system, but it is important to remember that each 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
  • Fixative pH
  • Ideal Fixation Temperature
  • Post-fixation treatment

Chemical vs physical fixation

Chemical fixatives crosslink 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 fixation procedure must be optimized to assure adequate fixation without altering the antigen 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 are usually fixed by drying, which removes the liquid from the sample and fixes the cells to 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 OCT compound, and then snap-frozen and stored in liquid nitrogen until they are sectioned. The example below provides an example of IHC staining in formalin fixed tissue. 

IHC was performed on a formalin fixed, paraffin embedded (FFPE) human colon cancer tissue section. To expose target proteins, heat-induced epitope retrieval (HIER) was performed using 10 mM sodium citrate buffer, pH 6, (e.g., 00-5000, AP-9003-125) for 20 min by heating at 95°C. Following antigen retrieval, cooling to room temperature and 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 activity was quenched 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 light microscope 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 bridge crosslinks

Formaldehyde and paraformaldehyde

Most commercial formaldehyde is prepared from paraformaldehyde (PFA, polymeric formaldehyde) dissolved in distilled/deionized water, with up to 10% (v/v) methanol added to stabilize the aqueous formaldehyde. Stabilization is important to prevent oxidation of the formaldehyde to formic acid and its eventual re-polymerization to paraformaldehyde. To avoid using methanol-stabilized formaldehyde 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 than true formaldehyde 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, which was 37 to 40% (w/v) formaldehyde, by diluting it 1:10 with phosphate buffer at neutral pH


Glutaraldehyde

Glutaraldehyde is a dialdehyde compound that reacts with amino and sulfhydryl groups 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 the tissue architecture. Tissues that have been fixed with a glutaraldehyde-based fixative must be treated or quenched with inert amine-containing molecules prior to the IHC staining because any free, unsaturated aldehyde groups that are available will react covalently with amine-containing moieties such as antibodies (Schiff base formation). The most efficient aldehyde blockers/quenchers are ethanolamine and lysine.

Other fixatives

Mercuric chloride-based fixatives are sometimes used as alternatives to aldehyde-based fixatives to overcome poor cytological preservation. These harsh fixatives work by reacting with amines, amides, amino acids like cysteine, and phosphate groups in proteins and nucleic acids.  The result is protein and nucleic acid coagulation, which can lead to undesirable tissue hardening. The benefits of using these fixatives are more intense IHC staining accompanied by the preservation of cytological detail allowing for easier morphological interpretation. These fixatives often include neutral salts containing zinc to maintain tonicity and they can be mixed with other fixatives to provide a balanced, less harsh 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 before IHC staining.  The main disadvantage of these mercury based fixatives is that they are highly toxic, corrosive, and they require special disposal procedures.  For this reason, they are not used frequently any more.

Precipitating fixatives include ethanol, methanol and acetone. These solvents precipitate and coagulate large protein molecules, thereby denaturing them, and can be good for cytological preservation. Such reagents can also permeabilize cells, which may be critical depending on the sample.  However, acetone, in particular, extracts lipids 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 fixatives are not appropriate for electron microscopy because they can cause severe tissue shrinkage. 

Diimidoester fixation using dimethyl suberimidate (DMS), an amine-reactive crosslinker, is a rarely-used alternative to aldehyde-based fixation (Hassel, J. et al., 1974). DMS is a homobifunctional reagent which crosslinks the α and ε-amino groups of proteins to each other. Diimidoesters are unique in that they create amidine linkages with the amines on the target molecules. As a result, DMS does not change the 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 include acrolein and glyoxal, which are similar to formaldehyde, and osmium tetroxide, which is particularly well-suited as a fixative prior to electron microscopy.  Other specialty fixative include carbodiimide 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.

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-buffered formalin (NBF)

Delicate tissue

Bouin's fixative

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's solution 
Helly solution

Nucleic acids

Carnoy's solution

Large protein antigens (e.g., immunoglobulins)

Ice-cold acetone (100%) or methanol (100%)

Ideal for electron microscopy 4% (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) Cytometry 8:235–239.
  4. Cowen T et al. (1985) Histochemistry 82:205–208.
  5. Mosiman VL et al. (1997) Cytometry 30:151–156.
  6. Romijn, Herms J. et al. (1999) J Histochem Cytochem 47:229–236.