Determine whether a direct or indirect method of staining is better for you depending on your particular experimental needs and the reagents available. Both methods allow for multi-color staining but have their own advantages and disadvantages.
Controls are extremely important – autofluorescence and non-specific binding of the primary or secondary antibody (for indirect detection) are 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 e.g. 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 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 and therefore a variety of methods should be investigated, for instance trying different percentages of Triton X-100 (0.1–0.25%).
During staining don't let your cells dry out at any point as this can introduce artifacts. This can be prevented by using a humidifying chamber.
After adding the fluorescent-labeled secondary antibodies make sure all subsequent incubation steps are performed in the dark to prevent quenching of the fluorescent dye/proteins.
When performing multi-color staining using indirect detection, minimize cross species reactivity and non-specific binding by using antibodies that have been pre-adsorbed against the host species of your primary and secondary antibodies.
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
Often ICC/IF images consist of only a few cells so it is important to make sure the image is typical and representative of the population.