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Immunohistochemistry (IHC) fixation immobilizes antigens while retaining cellular and subcellular structures. This protocol guides you through the process.
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Fixation immobilizes antigens while retaining cellular and subcellular structures. The fixation method used will depend on the sensitivity of the epitope and the antibodies themselves and may require some optimization.
Fixation can be done using crosslinking reagents such as paraformaldehyde. These are better at preserving cell structure but may reduce the antigenicity of some cell components as the crosslinking can obstruct antibody binding. Antigen retrieval techniques may be required, particularly if there is a long fixation incubation time or if a high percentage of crosslinking fixative is used.
For more information on IHC, see our complete guide
The correct fixative to use must be carefully considered for each IHC experiment as inappropriately fixed antigens may not be detected.
Fixing in formalin for more than 10–15 min will cross-link the proteins to the point where antigen retrieval may be required to ensure the antibody has free access to bind and detect the protein.
Ethanol and methanol will also permeabilize. Some epitopes are very sensitive to methanol as it can disrupt epitope structure. If this is occurring, try acetone instead if permeabilization is required.
Acetone will also permeabilize. No further permeabilization step is required.
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.
The ideal fixation time will depend on the size of the tissue block and the type of tissue, but fixation between 18–24 h seems to be suitable for most applications. Under-fixation can lead to edge staining, with strong signal on the edges of the section and no signal in the middle. Over-fixation can mask the epitope; antigen retrieval can help overcome this masking, but if the tissue has been fixed for a long period of time (i.e. over a weekend), there may be no signal even after antigen retrieval.
Perfusion fixation involves dissecting an animal and flushing 4% paraformaldehyde through its circulatory system via the heart. The tissue of interest can then be extracted and fixated further with immersion in the fixative.
If the tissue samples are fixed with an aldehyde fixative (paraformaldehyde, glutaraldehyde etc) for immunofluorescence detection, include 0.3 M glycine in the blocking buffer, before applying the primary antibody.
Glycine will bind free aldehyde groups that would otherwise bind the primary and secondary antibodies, leading to high background. Background due to free aldehyde groups is more likely to occur when the fixative is glutaraldehyde or paraformaldehyde.