ICC Protocols, Troubleshooting, & Scientific Resources
Secondary Antibody Handbook
Organelle Markers Guide
Immunocytochemistry (ICC) is an immunostaining technique that detects antigens in cultured or primary cells via light microscopy. Fluorochromes are the most common reporter molecules in microscopy and are standardly used in multicolor ICC experiments. Novus offers user-friendly resources for designing ICC/immunofluorescence (IF) experiments including a comprehensive overview of ICC, ICC/IF protocols, and a troubleshooting guide. Review our ICC/IF resources.
The example above depicts a multicolor ICC/IF scheme for indirect protein detection. HeLa cells were probed with anti-Ki67 (nuclear protein) and anti-Alpha-tubulin (microtubular protein) followed by detection with DyLightTM488 (green) and DyLightTM550 (red) conjugated secondary antibodies, respectively. Fixed cells were counterstained with the nuclear dye, DAPI (blue).
ICC/IF Primary Antibodies
The key to successful immunostaining is choosing an antibody that specifically binds its target antigen. Novus offers over 25,000 ICC/IF validated antibodies and numerous antibody-dye combinations to meet your multicolor ICC/IF needs. Our antibodies are 100% guaranteed, and you can try our antibodies in any untested species and applications using our Risk-Free Testing program.
Secondary Antibodies and Detection Reagents
In indirect IF detection, a fluorochrome labeled secondary antibody or a biotinylated secondary antibody paired with fluorochrome-conjugated streptavidin is required to detect the primary antibody-antigen complex. Compared to direct detection, the use of indirect detection enhances assay sensitivity and flexibility when designing ICC experiments. Learn more about the differences between direct and indirect detection in multicolor ICC/IF protocols.
Since variables at each step of ICC/IF protocol can contribute to background staining and false-positive or false-negative results, each experiment requires proper controls. Examples include peptide blocking controls, isotype controls, and cell line controls that either express or lack the target antigen. Learn more about ICC/IF Controls.
After the IF procedure, we recommend counterstaining cells with DNA intercalating dyes (e.g. DAPI) or other organelle binding dyes / antibodies (e.g. Phalloidin or GAPDH). This step is key for identifying cellular structures and individual cells. Learn more about counterstaining in ICC/IF.
Other ICC/IF Support Products
Besides antibodies and counterstains, Novus offers a variety of other support products for ICC/IF experiments, including blocking serum and mounting media.