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.
Considerations when choosing an ICC antibody
- 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.
- 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.
- 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.
ICC data examples
Each Invitrogen antibody that is indicated for ICC applications has undergone functional application testing. Here are some examples of that testing.
Detection of differential subcellular localization of Survivin during different phases of mitosis. Immunofluorescence analysis of Survivin using Survivin Monoclonal Antibody (1H5) (Cat. No. MA5-17035) shows Survivin in different mitotic phases of HeLa cells. There are low levels of Survivin at early prophase and an increase at late prophase. During metaphase, Survivin is localized in the centromeres, remains in the spindle midzone at anaphase, and localizes in the midbody between two daughter cells at telophase.
Membrane localization of Phospho-EGFR (Tyr1068) upon EGF treatment and inhibition of EGFR phosphorylation observed when treated with the antagonist, Afatinib. A-431 cells treated with 200 ng/mL of EGF for 10 minutes. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Phospho-EGFR (Tyr1068) Rabbit Polyclonal Antibody (Cat. No. 44-788G) and incubated overnight at 4°C and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 conjugate (Cat. No. A27034) (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade Gold Antifade Mountant with DAPI (Cat. No. S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Cat. No. R415). Panel d represents the merged image showing membrane localization. Panel e represents cells treated with antagonist, Afatinib (1 µM for 6 hrs) followed by EGF, showing no Phospho-EGFR staining. Panel f shows untreated cells with no signal. Panel g represents control cells with no primary antibody to assess background.
*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.