When beginning to plan an application in immunology, the selection of antibodies that can fulfil the intended function is important. Primary antibodies may be monoclonal or polyclonal. Other factors include the species they are raised in and any validation already available, such as KO-validation.
Another parameter to decide upon is the choice of staining – should it be direct or indirect? This depends upon the actual experiment and reagents on hand. Multiple colors in the stained specimen may be achieved in both cases, but other limitations and benefits specific for each must be considered.
The use of controls is vital to exclude misinterpretation of results because of autofluorescence and nonspecific interactions of the antibody (primary or secondary) when used for indirect detection. The controls used should be appropriate, such as:
Unstained sample: the sample without either primary or secondary antibodies should be used as control to determine basic autofluorescence levels
Secondary antibody: the isolated antibody should be used to detect nonspecific binding of the secondary antibody to any of the endogenous proteins in the cell
Only healthy cells should be used. Changes in the conditions of cell culture such as increased or too sparse cell density may alter the morphology of the cells in the final stage. Protein localization also varies in some cases with the degree of cell confluency. Thus cells should be grown on glass coverslips to achieve a confluency rate of 50%-60% at the stage of fixation.
When a cell or tissue is fixed, the cell morphology is preserved against change. However, this process may hide the epitope which is the binding target for the antibody, especially when monoclonal antibodies are used. Therefore, it may be skipped unless essential. The need for fixation is assessed by addition of antibody to unfixed as well as fixed cell specimens. If required, the fixation should be chosen depending on the specific epitope or antibody in the sample setup.
For intracellular proteins to undergo successful staining, the cells must allow antibodies to enter intracellular structures, which means their permeability should be artificially increased. Such permeabilization is accomplished by a variety of steps, chosen as per the antibody in use, in accordance with prior experimental verification. For instance, a series of experiments could be carried out using varying concentrations of the fixative Triton X-100 from 0.1% to 0.25%.
Acetone and methanol are examples of suitable permeabilization solvents for the detection of proteins inside the cell, but cannot be used with all antibodies.
Triton X-100 is an example of a detergent which can dissolve part of the nuclear membrane to allow antibodies to enter the nucleus and bind nuclear antigens. Its strong action can, however, lead to the breakdown of membrane proteins, especially if they are exposed to the detergent for too long. Detection of membrane proteins is therefore not an appropriate use for this chemical.
Suitable mild detergents which are acceptable alternatives to Triton X-100 include Tween 20 and saponin. These are solubilizers, and create pores in the cell membrane that allow the entry of antibodies into the cell while preserving the plasma membrane. They can be used to detect cytoplasmic or membrane antigens on the inner aspect of the plasma membrane, as well as soluble nuclear antigens.
An important precaution during staining is to avoid drying of the cell which leads to artefact formation. A simple preventive measure is the use of a humidification chamber.
After the secondary antibodies labeled with fluorescent tags are added, the rest of the cycle should be carried out in the dark to ensure that the fluorescent proteins/dyes are not quenched.
When indirect detection is being performed with multi-color antibodies, cross-species reactivity should be minimized, as well as nonspecific interactions, by ensuring that the antibodies used have already undergone pre-adsorption against the species in which the primary and secondary antibodies were raised.
Other ways to minimize nonspecific binding include:
- Using blocking serum from the same species in which the secondary antibody was raised
- Reducing the amount of antibody
- Reducing the incubation time
- Addition of 0.1M glycine to quench any aldehyde left in the solution after fixing with formaldehyde
If protein targets in a cell are to be studied with regard to their distribution or localization within the cell, organelle-specific markers may be targeted using appropriate antibodies to produce counterstaining. Such markers may include DRAQ5/7 for DNA or nucleus, tubulin for the plasma membrane, and TGN46 for Golgi apparatus.
Since images acquired in ICC/IF typically contain only a small number of cells, it must be ensured that they represent the typical cell distribution and cell type of the population.
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