Just when you think that you’ve found the perfect fixative for your tissue, there’s something else to consider- your fixative may have just masked all the antigen sites your lovely new antibody was going to bind to!
Have you just masked your antigen?
As I mentioned in one of my previous articles, the fixation technique you chose usually creates cross-links within proteins; this is one of the ways in which tissue is stabilized. This can alter the biochemistry of the protein such that the antigen of interest is masked and will no longer be recognised by your primary antibody.
Don’t worry- help is at hand!
Antigen masking can be caused not only by cross-linking the amino acids within the epitope, but also by cross-linking unrelated peptides at or near an epitope, altering the conformation of an epitope, or altering the electrostatic charge of the antigen. Because antigen-antibody recognition relies upon protein structure, it is important to try to restore the natural 3-D structure. Don’t worry- there are a number of techniques available to get past this.
Remove your mask!
Some antibodies do not require any kind of unmasking but if yours does, don’t panic- it’s only a short add-on to your usual protocol. The term antigen retrieval refers to any technique in which the masking of an epitope is reversed and epitope-antibody binding is restored. The type of antigen retrieval depends on many variables which include the target antigen, the antibody used, tissue type and the method of fixation used to preserve your tissue. For example, if you are using a polyclonal antibody, which recognizes multiple epitopes, it is less likely to require antigen retrieval, but it’s also likely to have a little extra background. If you used a fixation technique such as ethanol, then antigen retrieval is not recommended as the fixation may not be strong enough to handle retrieval conditions.
Heat or enzymes- you choose
There are multiple techniques to restore the immunoreactivity of an epitope and these generally fall into two main categories; (a) enzyme-induced antigen retrieval and (b) heat-induced antigen retrieval. Also, within each category there are a number of different ways to do the retrieval.
In the enzyme-induced methods, enzymes including Proteinase K, Trypsin, and Pepsin can be used to restore the binding of an antibody to its epitope. The mechanism of action is thought to be the cleavage of peptides that may be masking the epitope. The main enzyme-induced methods, which require a 10-20 minute incubation at 37?C, are:
- Proteinase K (20 g/ml in TE buffer, pH 8)
- Trypsin (0.5% in dH20)
- Pepsin (0.1% in 10 mM HCl)
- Pronase (0.5 or 0.1% in dH20)
- Protease (0.5% in dH20)
Depending on whichever method you choose, each one must be optimized for different tissue types and antibodies, but each of these methods slot in before blocking and after the dewaxing steps. The disadvantages of these methods are the low success rate for restoring immunoreactivity and the potential for destroying both tissue morphology and the antigen of interest.
If you find the enzyme-induced way a little too harsh, or that it simply doesn’t work for you, then you can try the heat-induced method. This method is believed to reverse some cross-links and allows for restoration of secondary or tertiary structure of the epitope. It is, in essence a re-naturation of the structure of fixed proteins through a series of conformational changes.
This may include the possible hydrolysis (breaking) of formalin-induced crosslinks with the entire process being driven by thermal energy from the heat source. As with the enzyme-induced methods, the heat-induced methods require optimization. These methods, which require 10-40 minutes in a steamer, microwave (five minute increments) or water bath at 95-100?C followed by 20 minutes cooling at room temperature are:
- Citrate buffer (10 mM Sodium Citrate, 0.05% Tween 20, pH 6)
- Citrate-EDTA buffer (10 mM Sodium Citrate, 2 mM EDTA, 0.05% Tween 20, pH 6.2)
- EDTA (1 mM EDTA, 0.05% Tween 20, pH 8)
- Tris-EDTA (10 mM Tris Base, 1 mM EDTA, 0.05% Tween 20, pH 9)
- Glycine-EDTA (50 mM Glycine-HCl, 0.01% EDTA, pH 3.5)
In general, the heat-induced methods are more widely used, probably because they are more successful. If you are just beginning antigen retrieval then a good place to start out would be the tried and tested method of citrate buffer retrieval. Of course, if it doesn’t work, then you have plenty to try afterwards.