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Fixation Strategies and Formulations used in IHC staining

For immunohistochemical analysis, it is essential that the morphology of the tissues and cells are retained and the antigenic sites accessible.

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Overview of Immunohistochemistry
(includes links to individual pages on all aspects and stages of IHC)

Introduction

Fixation plays four critical roles in immunohistochemistry (IHC):

  • Stabilize cell morphology and tissue architecture
  • Disable proteolytic enzymes
  • Strengthen samples to withstand further processing and staining
  • Protect samples against microbial contamination and decomposition

The right fixation method requires optimization based on the application and the target antigen to be stained. Methods of fixation include:

  • Perfusion: Tissues can be perfused with fixative following exsanguination and saline perfusion to allow rapid fixation of deep tissue.
  • Immersion: Samples are immersed in fixative to allow diffusion 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 sensitive for chemical fixation or exposure to organic solvents during deparaffinization can be embedded in cryoprotective embedding medium, such as OCT, and then snap-frozen and stored in liquid nitrogen.
  • Drying: Blood smears for ICC 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 epitope, it may destroy other epitopes, even if they are on the same antigen. The guidelines on this page are helpful in determining the appropriate fixative for a particular system, but each antigen is unique. Therefore, the following considerations should be addressed when choosing a fixative:

  • Type of fixative (formaldehyde, glutaraldehyde, etc.)
  • Rate of penetration and fixation
  • Fixative concentration
  • pH
  • 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. Each fixative procedure must therefore be optimized to balance 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 fixed by drying, which removes the liquid component of the sample and fixes the whole 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, and then snap-frozen and stored in liquid nitrogen until sectioned.

 
fluorescent fluorescence vimentin lamin B1 IHC immunohistochemistry horseradish peroxidase DAB
Chromogenic IHC detection of ezrin in colon carcinoma tissue. Human colon carcinoma tissue samples were fixed with 4% paraformaldehyde and incubating with anti-ezrin antibody followed by HRP-Conjugated Goat Anti-Mouse Secondary Antibody. The signal was then developed with Pierce Metal-Enhanced DAB.
 

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 bridges.

Formaldehyde and Paraformaldehyde

Most commercial formaldehyde is prepared with paraformaldehyde (polymeric formaldehyde) dissolved in distilled/deionized water, with methanol added to stabilize the aqueous formaldehyde. Solution stabilization is important to prevent oxidation to formic acid and the eventual repolymerization to paraformaldehyde, and therefore commercial formaldehyde may contain up to 10% methanol. To avoid using methanol-contaminated 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 formaldehyde solutions. For example, 10% neutral-buffered formalin (NBF or simply formalin) is really a 4% formaldehyde solution; the basis for this difference is that historically, formalin was prepared with commercial-grade stock formaldehyde, which is 37 to 40% formaldehyde, by diluting it 1:10 in phosphate buffer.

 

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>Methanol-free Formaldehyde

Glutaraldehyde

Glutaraldehyde is a dialdehyde that reacts with amino groups, sulfhydryl groups and possibly with aromatic ring structures. Fixatives containing glutaraldehyde are stronger protein crosslinkers than formaldehyde. They penetrate tissue more slowly, causing extraction of soluble antigens and modification of the structure. Tissues that have been fixed with a glutaraldehyde-based fixative must be treated with inert amine-containing molecules prior to the immunoassay because free, unsaturated aldehyde groups are available to covalently link amine-containing moieties such as antibodies. The most efficient aldehyde blockers are ethanolamine and lysine.

 

Other Fixatives

Mercuric chloride-based fixatives are used as an alternative to formaldehyde-based fixatives to overcome poor cytological preservation. These fixatives work by additive and coagulative properties. The major advantages of using these fixatives include good penetration resulting in more intense immunostaining and the preservation of cytological detail allowing for easier morphological interpretation. These fixatives often contain neutral salt to maintain tonicity and can be mixed with other fixatives to provide a balanced solution. Mercuric chloride-based fixatives include Helly and Zenker's Solution. Sections must be cleared of mercury deposits before immunostaining.

Precipitating fixatives include ethanol, methanol and acetone. They precipitate large protein molecules and are good for cytological preservation. These fixation reagents can also permeabilize cells, which may be critical depending on the sample. They are not good for electron microscopy, though, because they cause tissue shrinkage.

Diimidoester fixation using dimethyl suberimidate (DMS), an amine-reactive crosslinker, is an alternative to aldehyde-based fixation (Hassel, J. et al., 1974). DMS is a bifunctional reagent with the α and ε-amino groups of proteins. Diimidoesters are unique in that they carry an amido group next to the functional groups on the molecules. As a result, DMS does not affect 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.

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>Dimethyl Suberimidate (DMS)

Fixative Formulations for Specific Applications

While histochemistry and histopathology texts describe in detail a legion of different fixatives and their results 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.
Antigen Fixative
Most proteins, peptides and enzymes of low molecular weight 4% Paraformaldehyde
4% Paraformaldehyde-1% Glutaraldehyde
10% Neutral-Buffered Formalin (NBF)
Delicate tissue Bouin's Fixative
Small molecules such as amino acids 4% Paraformaldehyde-1% Glutaraldehyde
Blood-forming organs (liver, spleen, bone marrow); connective tissue Zenker's Solution
Helly Solution
Nucleic acids Carnoy's Solution
Large protein antigens (immunoglobulin) Ice-Cold Acetone or Methanol (100%)
Ideal for electron microscopy 4% Paraformaldehyde-1% Glutaraldehyde
 
Common fixative formulations and notes on their storage and use.
4% Paraformaldehyde in 0.1M Phosphate Buffer

Mix together:
   • NaH2PO4, 3.2g
   • Na2HPO4, 10.9g
   • Distilled water, 1000mL
Adjust pH to 7.4
Then add:
   • Paraformaldehyde, 40g

Heat mixture to 60°C while stirring and add 1-2 drops of 1N NaOH to help paraformaldehyde dissolve. Cool and filter the solution.
4% Paraformaldehyde-1% Glutaraldehyde in 0.1M Phosphate Buffer
Prepare 4% paraformaldehyde in
0.1M phosphate buffer, as above.
Then add:
   • Glutaraldehyde, 20mL
 
Bouin's Fixative

Mix together:
   • Saturated aqueous picric acid, 750mL
   • 40% formaldehyde, 250mL
   • Glacial acetic acid, 50mL

Store at room temperature
10% Neutral-Buffered Formalin

Mix together:
   • Na2HPO4, anhydrous, 6.5g
   • NaH2PO4•H20, 4g
   • Distilled water, 900mL
Adjust pH to 7.4
Then add:
   • 40% formaldehyde, 100mL

Store at 4°C
Carnoy's Solution

Mix together:
   • Ethanol (absolute), 60mL
   • Chloroform, 30mL
   • Acetic acid, glacial, 10mL

 
Helly Solution

Mix together:
   • Mercuric chloride, 5g
   • Potassium dichromate, 2.5g
   • Distilled water, 100mL
   • 40% formaldehyde, 5mL

Heat, cool, filter in brown bottle. Wash sample for 24 hours with distilled water after fixation. Never use metal forceps to handle tissue.
Zenker's Solution

Mix together:
   • Mercuric chloride, 5g
   • Potassium dichromate, 2.5g
   • Distilled water, 100mL
   • Acetic acid, glacial, 5mL †

Heat, cool, filter in brown bottle. Wash sample for 24 hours with distilled water after fixation. Never use metal forceps to handle tissue.
Precipitating Solutions

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

Fix for 5-10 minutes at room temperature. Great for fixing AND permeabilizing, if needed.

†Add component right before use
 

References

  1. Hassel, J. and Hand, A.R. (1974). J. Histochem. Cytochem. 22 229-239.
 
Written and/or reviewed by Jared Snider, Ph.D.

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