All tags Primary antibodies Tips for successful immunocytochemistry/immunofluorescence experiments

Tips for successful immunocytochemistry/immunofluorescence experiments


Technical hints and tips to ensure optimal performance of your ICC/IF experiments.


Planning your experiment

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. 


Experimental controls

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:

  • 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 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.


Fixation/permeabilization

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%).

  • 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 also partially dissolve the nuclear membrane and are therefore 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 and are therefore 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, and are also suitable for soluble nuclear antigens.


Staining

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:

  • 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


Co-localization/counterstaining

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


Image acquisition

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.



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