Sample fixation is a required and crucial step for every successful IHC/ICC experiment. Appropriate fixation of samples provides the following benefits during the tissue preparation process:
- Preserves morphology
- Prevents tissue degradation
- Ensures antigenicity
- Protects cells from damage
Choosing which fixing solution to use depends on your sample type and antigen. Since there is no standard fixing solution for all samples, we recommend testing to determine which specific type of solution will be most appropriate and effective for antigen immobilization in your sample.
As an example, compare the morphologies demonstrated below using different fixatives, both photographed at the same magnification.
On the left: A paraffin section of the small intestine mucosa that has been fixed in neutral buffered formalin, a cross-linking fixative. Nuclear and cytoplasmic preservation is satisfactory but some cellular shrinkage is present.
On the right: A paraffin section of the small intestine mucosa that has been fixed in 95% ethanol, a denaturing fixative. While nuclear preservation is fair, there is substantial shrinkage of cytoplasmic and extracellular elements.
Several fixing solutions are available for use and should be chosen based on the sample type or antigen studied in the experiment. Below are the 3 different categories of fixatives:
Aldehyde fixatives are di-functional cross-linking agents, which are widely used due to their strong penetrability, low contractibility, and low background. They help keep the cross-linking between tissues and maintain the antigen.
Examples include: Formaldehyde/Formalin (most common fixative), Paraformaldehyde, Glutaraldehyde, Bouin’s solution, Zamboni’s solution.
What is the difference between formaldehyde, formalin, and paraformaldehyde?
Formaldehyde and formalin are often referred to interchangeably. They are similar, but their chemical compositions are in fact different. Formalin (aka NBF) is a saturated water solution consisting of 37 to 40% (w/v) formaldehyde, which is diluted with a phosphate buffer to decelerate polymerization to formaldehyde. As a result, formalin’s fixing ability is decreased.
Paraformaldehyde (or polyoxymethylene) is polymerized formaldehyde powder. Dissolving the powder in hot distilled water and adding 10% (v/v) methanol will produce the stabilized formaldehyde solution.
Acetone and alcohol play the role of precipitating sugars and fat in addition to maintaining immunologic competence. Acetone is often chosen for unfixed, snap-frozen tissues and cytological smears due to its strong penetrability and dehydration property. Methanol and ethanol are the most common alcohols selected for cell and tissue fixation because of the similarities of their molecular structures with water, which enables them to substitute water molecules in tissues by competing for protein hydrogen bonds.
In cases where aldehyde or precipitating fixatives are not optimal choices for the sample type or antigen, non-aldehyde fixatives have been used as alternatives.
Mercuric chloride-based fixatives: These fixatives provide more intense IHC staining while preserving cytological details more effectively than aldehydes. However, this alternative also results in tissue hardening, which may be troublesome.
Diimidoester fixation: This fixation method utilizes dimethyl suberimidate (DMS), which offers the advantages of retaining antigen immunoreactivity and eliminates the need to block aldehyde groups.
Other non-aldehyde fixatives include carbodiimide, dimethylacetamide, para-benzoquinone, etc.
Note: In some situations, these non-aldehyde fixation agents can be mixed with other tissue fixatives for a more optimal solution.