Immunofluorescence (IF) is a detection technique that uses fluorochrome-labeled antibodies to visualize targets. IF is commonly used together with immunocytochemistry (ICC) or immunohistochemistry (IHC). Antigens in the sample, tissue, or cells, can be visualized through direct or indirect detection. Direct detection involves use of fluorochrome-conjugated primary antibodies. Whereas indirect detection involves an unlabeled primary antibody followed by a fluorochrome-conjugated secondary antibody. Optimal labeling and detection methods are critical for achieving high signal-to-noise ratios with immunolabeled samples.

All Antibodies for use in IF

Considerations when choosing an IF antibody

  1. Whether it will work in ICC/IHC. Not all antibodies work for all applications. An antibody for IF should also work for ICC or IHC. After ascertaining that the antibody does in fact for these applications, it can be followed up with either direct or indirect immunofluorescence detection methods.
  2. Whether it is specific. Specificity of antibodies 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 the said 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. Different fluorochrome-labeled antibodies can be combined in a multiplex ICC/IHC. Multiplexing allows for 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 for immunofluorescent detection. The key requirement is that the specific epitope of interest be exposed. But, the advantages and disadvantages of using a monoclonal versus a polyclonal antibody should be assessed depending on the research needs. An advantage of using a monoclonal antibody is that, generally, it is more specific. However, this is associated with a higher likelihood that the one epitope the monoclonal antibody recognizes is buried. Unless monoclonal antibodies are specifically screened or designed for use in IF, polyclonal antibodies would be a good alternative for recognizing target proteins. Polyclonal antibodies recognize multiple epitopes of the targets. However, they are more likely to be cross-reactive.

IF data examples

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

Detection of SNAP25 in PC12 cells differentiated to neuronal phenotype. For immunofluorescence analysis, PC12 cells were fixed and permeabilized for detection of endogenous SNAP25 using SNAP25 Recombinant Rabbit Monoclonal Antibody (Cat. No. 701991, 5 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 conjugate (Cat. No. A27034, 1:2000). Nuclei (blue) is stained using SlowFade Gold Antifade Mountant with DAPI (Cat. No. S36938) and cytoskeletal F-actin (red) staining using Rhodamine Phalloidin (Cat. No. R415, 1:300) Panel a-d shows representative un-treated cells that were stained for detection and localization of SNAP25 protein (green) with reduced signal compared to panel e-h, clearly demonstrating enhanced cytoplasmic and membrane localization of SNAP-25 in PC12 cells differentiated to neuronal phenotype with NGF (200 ng/mL 5 days). The images were captured at 60X magnification.

For Research Use Only. Not for use in diagnostic procedures.