Immunostaining is a complex technology and subject to several technical obstacles, which could mean that after a lot of time and effort the result is either no signal or one which is impossible to interpret, instead of a clear and well defined set of images. But even this is not the worst scenario.
Worst of all, the outcome could be something that looks very specific, but is misleading or plain wrong.
Nancy Kedersha, Instructor of Medicine at Harvard Medical School, and Director of the Confocal Microscopy Core at Brigham and Women’s Hospital
Nancy’s expert recommendations are the basis of this article on immunostaining, as she shares her insights on what makes immunostaining experiments on cultured cells most likely to succeed.
The Right Environment for the Technique
The first step in setting up a successful immunostaining test is using the right buffers. Antibody proteins are complex molecules, designed to work in the blood, which contains a high sodium ion concentration. This is the reason why phosphate buffered saline (PBS) is highly recommended for staining the cell surface. However, since the interior of the cell is dominated by the presence of potassium ions, it is important to approximate these conditions as far as possible.
This is where the buffer system becomes so important, for after all, it is used while the cells are being incubated and also during the cell washes after fixation and permeabilization are carried out. Its presence can alter the intensity of the signal to a great extent.
Some buffers were meant to be used in cells which had not been fixed but had been permeabilized, and were to be used in assays to determine function. These include PHEM and CSK. On the other hand, these can be substituted for PBS in assays of cells which have undergone fixation and permeabilization, especially when dealing with antigens present in the cytoskeleton or cytoplasm.
When signal intensity is considered, experience tells us that staining is markedly improved in about 10% of cell antigens when PHEM is used as the buffer rather than PBS, while 50% show moderate enhancement and 30% do not show any significant difference.
The last 10% show much better staining with PBS than with PHEM. Thus it is wise to use both PBS and PHEM when performing immunostaining on an antibody being used for the first time. Other buffers that could be tried are listed below:
The Optimum Fixative
A successful immunostaining experiment demands that the antigen-antibody combination be paired with the right fixative. Immunostaining protocols require the right buffer, as already discussed, but also the cells must be maintained in as pristine a condition as possible while incubations and washes are being performed.
Fixation is a vital step in this respect as it is essential to keep the structure of the cells intact, as much as possible. After this is complete, the rest of the staining protocol can be carried out without fear of damaging the cell, especially the target protein. However, antigenic sites are disrupted by fixation, which means that the same fixative may not maintain all antigen-antibody combinations in good working order even if it has shown excellent results with one of them.
If a new protein is being stained, and particularly if its location is somewhat uncertain, or if the antibody being used is unfamiliar, the fixative-buffer combination should be varied several times to determine which will provide the best mix of antibody binding with minimal structural disruption.
Fixatives in common use fall into two categories: aldehydes and organic solvents. Neither class is indisputably superior, and this is why many methods should be tried out when attempting to work with a new antibody or novel target.
Aldehyde fixatives are usually formaldehyde or glutaraldehyde, which create crosslinking between cytoskeletal proteins and other components. This allows membrane-bound antigens and antigens on the cytoskeleton to be double-labeled, and is the first choice in this situation.
The downside is the chemical change it induces in the proteins which is capable of destroying the antigens. Cell fixation is not usually associated with antigen destruction as in most cases it is accomplished by the use of 2-4% paraformaldehyde for 10-20 minutes. The problem is that aldehyde fixation can only be accomplished by exposing the tissues to the chemical for prolonged periods, and this results in a change in the structure of the protein.
This is why it is always recommended that fixation times should be as short as is feasible, for both cells and tissues. Another potential obstacle is the occurrence of autofluorescence following the use of an aldehyde for fixation, via the creation of fluorescent products by the reaction of amines and proteins. This means an additional step called quenching is necessary.
Organic solvents include methanol, ethanol and acetone. These do not react with the protein of interest by covalent reaction, but cause the dissolved protein to precipitate, keeping the protein shell intact but reducing the solubility of the protein such that the cells are flattened.
The issue here is that it becomes more difficult to gain entry to the mitochondria and nucleus, while also removing proteins which are linked to (unprotected) lipids. In some cases this is all to the good, however, as some antibodies bind only to antigens buried inside the protein structure. One important example of this would be monoclonal antibodies that bind to a single epitope. This is recommended to preserve cells and to stain cytoskeletal structures.
Formaldehyde: A Brief Description
Formaldehyde, paraformaldehyde and formalin are often mistaken for each other. Among these three, the simplest aldehyde is formaldehyde, CH2O. Paraformaldehyde is produced by the polymerization of formaldehyde, and is available as a powder that must be heated to dissolving point in a fume hood.
The solution of paraformaldehyde takes a significantly long period to enter the cell, and permeabilization proceeds too slowly for antibodies to be able to penetrate to the cell interior. This means that a permeabilization step is additionally required.
Formalin is a liquid composed of 37% formaldehyde with the addition of 10-15% methanol in order to inhibit paraformaldehyde formation. The presence of the methanol is not to be ignored as it is an organic solvent in its own right.
Thus if a protocol includes the use of 10% formalin, it should be noted that this is equivalent to the use of 4% formaldehyde plus sufficient methanol to cause partial permeabilization. However, without exposing the cells to the formalin for a long time, consistent permeabilization cannot be predicted.
The Need for Fixation
Why is cell fixation needed at all? After all, it is known to cause damage to or to mask some antigens, which means the signal intensity following immunostaining will be less. Thus it is all right to try the protocol with some modifications, such as adding antibody before fixation, or cutting out the fixation step completely. This is especially to be tried when trying to stain cell surface proteins which are exposed, as seen in Figure 1.1.
In such a case the antibody should be added to the culture medium just to verify the presence of the target antigen on the surface. However, every step should be taken with care, remembering that there are many molecules on the cell surface which are being passed into the cell at any given time, or which will enter the cell interior if crosslinking happens. This is a strong possibility when antibodies, which have two binding sites, are added to the solution. Another factor affecting this step is that some of the antigens which take part in adhesion between cells may suffer impairment of function following their binding by antibodies.
A secondary permeabilization step is often part of several protocols which use formalin or formaldehyde as the fixative. This may be done using detergents or organic solvents. If the first are preferred, the choice lies between mild to moderate detergents, saponin or digitonin, 0.1-0.5% Triton-X100 and NP40.
Organic solvents include methanol and acetone. Of course, permeabilization is not required as an additional step if cell fixation is done using organic solvents. The figure below shows the effectiveness of permeabilization.
Selection of Antibodies
Antibodies are of various kinds. Some bind to only a minute region of a molecule targeted by them, such as monoclonal and peptide-specific antibodies. These are produced by exposing the cell to only a small region of a very large molecule. There are a host of commercial antibodies that are of this type, but not all antibodies do so.
Some are raised from antigens which comprise the whole protein. These are polyvalent antibodies, and comprise a mixture of many antibodies that originate from many clones of immune cells, each antibody being specific against a small part of the whole large antigen, as shown in Figure 3.
Generally speaking, the polyvalency of an antibody increases its affinity and such antibodies are especially important in antibody localization studies, as well as in immunoprecipitation and immunoblotting. Experience shows, however, that monoclonal and peptide-specific antibodies also have their own peculiar place.
When an antibody reacts to a whole protein, it may still react selectively to one part of the whole molecule. On example is of a tetrameric heterodimer, a molecule made up of four units of different kinds. In such a case, it may happen that two subunits of this macromolecule are not the components recognized by the polyvalent antibody.
Antibodies reacting with a single epitope with high affinity are also used to detect phosphorylation sites, O-GlcNAc modifications and some epitopes with specific conformations.
Thus polyvalent and single-epitope antibodies are best used in combination if the aim is to both identify a given protein and to study its functional state.
Points to Remember
When an antibody or a cell is subjected to immunostaining procedures, a failure may occur due to the protocol rather than any property of the cell or antibody. Especially when cell surface antigens are to be studied, a strict protocol works well.
But when the antigen is inside the cell, the typical need is to detect small molecules on cell structures in the interior of the cell, using the much larger antibody molecules. This requires permeabilization, or the production of cell membrane holes, of sufficient size for antibody penetration to occur to the interior, while simultaneously taking care to prevent adverse effects due to the presence of these molecules inside the cell as fully as possible.
Thus a single-size solution is just not available for immunostaining of antigen targets inside a cell, and this is more obviously a challenge when the protein of interest is being stained for the first time. The best thing is to experiment with different methods at hand, discovering their limitations and advantages relative to each other.
Each antibody and antigen encountered for the first time must be tested in a variety of different ways to find the best immunostaining method for each separate combination of antigen and antibody. The tips covered here are helpful to achieve good immunostaining without too much hassle, and should be remembered from the outset.
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