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Technical hints and tips to ensure great results from your immunofluorescence (ICC/IF) experiments.
Controls are extremely important – autofluorescence and non-specific binding of the primary or secondary antibody (for indirect detection) is often misinterpreted as genuine staining. It is therefore important to ensure the correct controls are used, including the following:
Ensure your cells are healthy as differences in culture conditions eg cell density, can affect cell morphology. A good target to aim for is to grow cells on glass coverslips to 50-60% confluency on the day of fixation. If cells are too densely or sparsely packed then normal cell structure can be affected. Additionally, some proteins exhibit differences in localization depending on cell confluency.
Cell or tissue fixation preserves cellular morphology and structure but may mask the epitope that the antibody binds to (this is especially true for monoclonal antibodies). To see if fixation is required for your sample try adding the antibody to cells which have and have not been fixed. The optimum fixative will vary depending on your target/antibody used.
Staining intracellular proteins requires cell permeabilization to allow antibodies access to the intracellular components. For each antibody, the optimal permeabilization step will be different so a variety of methods should be investigated, for instance trying different percentages of Triton X-100 (0.1–0.25%).
Non-specific staining may be reduced by:
To examine the distribution or any changes in subcellular localization of protein targets, antibodies specific for an organelle marker can be used for co-localization/counterstaining. Common markers include tubulin (plasma membrane), TGN46 (Golgi) and DRAQ5/7 (DNA/nuclei).
View our organelle markers antibodies
ICC/IF images often consist of only a few cells so it is important to make sure the image is typical and representative of the population.