Antibodies for Immunocytochemistry (ICC)

Immunocytochemistry (ICC) combines immunological and biochemical techniques to image discrete components in cells (grown in a monolayer or in suspension) by using appropriately labeled antibodies to bind specifically to their target antigens in situ. ICC enables the visualization of the subcellular localization of the target antigen and gives information on its relative abundance. ICC can also be used to visualize the physiological behavior of a protein of interest in the cellular context in response to a cell treatment. Traditional ICC relies on the same enzyme reactions as immunohistochemistry (IHC) where the staining process exploits enzymes that catalyze the deposition of a colored staining product at antigenic sites within the sample.

However, it is much more common to see ICC used generically to describe immunofluorescence (IF) staining cells. This procedure is commonly accomplished either by using fluorophore-conjugated primary antibodies raised against a specific protein, or by first labeling with primary antibodies followed by secondary antibody detection. Optimal labeling and detection methods are critical for achieving high signal-to-noise ratios with immunolabeled cells.

All Antibodies for use in ICC

1. Whether it will work in ICC. Not all antibodies work for all applications. If the antibody has been validated* for ICC, then the manufacturer’s guidelines or any published references can be used as guidance for the experiment. Other methods that require the antibody to recognize the target in a native state, such as immunofluorescence (IF) and immunohistochemistry (IHC), are also good indicators.
2. Whether it is specific. Specificity of the antibody has been a growing and understandable concern. Antibody specificity becomes more important when studying post-translational modifications or specific isoforms of the protein. Invitrogen antibodies undergo a two-part testing approach: functional application validation and targeted specificity verification. Functional application validation provides information on whether the antibody works in an application. Target specificity verification ensures the antibody is recognizing the target protein of interest.
3. Selection of primary antibody based on host. More than one protein can be detected through multiplexing. This way target co-localization studies can be carried out. Primary antibodies from different species must be used to prevent secondary antibody cross-reactivity and improve specificity.

Both monoclonal and polyclonal antibodies can work in ICC. The key requirement is that the specific epitope of interest be exposed. One of the advantages of using a monoclonal antibody is that generally it is more specific. However, monoclonal antibodies are associated with a higher likelihood that the one epitope it recognizes is buried. Unless monoclonal antibodies are specifically screened or designed for use in ICC, polyclonal antibodies may be better candidates for recognizing target proteins. Polyclonal antibodies recognize multiple epitopes of the target, but concurrently, are more likely to be cross-reactive.

Each Invitrogen antibody that is indicated for ICC applications has undergone functional application testing. Here are some examples of that testing.

*The use or any variation of the word “validation” refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical or diagnostic uses.