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.
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.
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.
Immunofluorescence analysis of iPSC differentiated to intestinal organoids. For immunofluorescence analysis, iPSC differentiated to intestinal organoids were fixed and permeabilized for detection of endogenous SOX9 using SOX9 Recombinant Rabbit monoclonal Antibody (Cat. No. 702016, 1:100 dilution) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 conjugate (Cat. No. A27034, 1:2000). Panel b) shows representative cells that were stained for detection and localization of SOX9 protein (green) in the nucleus of cells lining the lumen of intestinal organoids in comparison to iPSC (a).
*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.
For Research Use Only. Not for use in diagnostic procedures.