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Fixation immobilizes antigens while retaining cellular and subcellular structure. 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.
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 (ie over a weekend), there may be no signal even after antigen retrieval.
Adequate fixation is often obtained by immersion of small tissue pieces into the fixative solution. However, a more rapid and uniform fixation is usually obtained if the fixative solution is perfused via the vascular system, either through the heart or through the abdominal aorta. The following procedures provide fixation of most rat organs with 4% paraformaldehyde.
Materials for perfusion fixation
Perfusion fixation through the heart
Perfusion fixation through the abdominal aorta
Solution coming out of animal’s orifices – the needle is inserted incorrectly.
Try to get the needle up the left atrium so that the solution does not flow into the lungs. Be careful not to insert the needle too far up as there is a risk of puncturing an internal wall. Getting the right position may require some practice, so try moving the needle around a bit until the solution stops flowing from the animal’s nostrils or mouth.
Fixation taking too long – the fixative solution is not circulating properly.
Make sure that you have properly clamped the needle at the point of entry and that there is no leakage/puncture to the heart. This can be easily done by dabbing dry the area around the heart to see if the fixative is leaking out. Also, try repositioning the needle insert.
Punctured heart – the perfusion fixation may not be complete and the sample organ/tissue may not be properly fixed.
Try to clamp the puncture wound close. Continue with the perfusion fixation but make sure to fix the organ in the same fixation for more than two hours after excising it. In this case, it would be best to do immersion-fixation at 4°C, overnight.
If your sample organ is a testis, after excising it, try injecting some of the fixative into the apical regions, right under the tunica albuginea layer, so that the seminiferous tubules inside will be fixed. The testis will bloat up a bit and feel hard if it is has been injected correctly.
If you are able to locate a vein in your sample organ, you can manually try to inject (using a syringe) the fixative solution into the vein.
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.