Technical hints and tips to ensure great results from your immunofluorescence (ICC/IF) experiments.
Choosing your antibodies
It's important to be sure that your antibodies are suited to your intended application before you start your experiment. Consider the type of primary antibody you are using ie monoclonal vs polyclonal, the host species as well as any shown validation. For more information on how to choose the best antibody for your experiments, look through our antibody guide pages or read why KO-validated antibodies are so important.
Determine whether a direct or indirect method of staining is better for you depending on your experimental needs and the reagents available. Both methods allow for multi-color staining but have their own advantages and disadvantages.
Chose the secondary antibody specific to the species primary antibody was raised on. If you use a primary antibody raised in rabbit, you will need an anti-rabbit secondary antibody raised in a host species other than rabbit (e.g. Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150073).
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:
Unstained sample (no primary or secondary antibodies) – to understand the levels of autofluorescence
Secondary antibody alone – to determine the possibility of the secondary antibody binding non-specifically to the endogenous cell proteins
Preparing your cells
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%).
Solvents such as acetone and methanol are suitable for permeabilization when detecting intracellular protein; however, they are not compatible with all antibodies.
Detergents such as Triton X-100 will partially dissolve the nuclear membrane and are suitable when access to nuclear antigens is required. However, they are harsh detergents that can disrupt membrane proteins, especially if left on for too long. This means they are not suitable for detecting membrane proteins.
Tween 20 and Saponin are much milder membrane solubilizers. They will create pores large enough for antibodies to pass through without dissolving the plasma membrane. They are suitable for antigens in the cytoplasm or the cytoplasmic face of the plasma membrane. They are also suitable for soluble nuclear antigens.
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. This prevents 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:
Blocking with serum from the host species of your secondary antibody
Using less antibody and/or decreasing the incubation times
Quenching residual aldehydes following formaldehyde fixation using 0.1M glycine
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).