Detection Methods for ICC IF

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Detection Methods

In fluorescence-based immunodetection, antibodies are conjugated to fluorochromes which emit light upon excitation with light at a shorter wavelength. While detection typically involves either a labeled primary or secondary antibody, using a biotinylated antibody followed by fluorochrome-labeled streptavidin improves assay sensitivity.

Direct and Indirect Detection

Direct detection in multicolor ICC/IF uses primary antibodies that are conjugated to a fluorochrome. In indirect detection, the primary antibody targeting the molecule of interest is unlabeled and a labeled secondary antibody is directed against the constant region of the primary antibody.

Direct and indirect detection

Which detection method is better – direct or indirect?

The optimal detection method may vary from experiment to experiment. The factors and considerations listed in the table below can help determine which method is best suited for your experiment.

	Direct detection method	Indirect detection method
Sensitivity	The direct method of detection is less sensitive because the amplification of signal afforded by the secondary antibody is lost.	The indirect method of detection is highly sensitive as more than one secondary antibody can bind to a single primary antibody molecule.
Assay Time	The direct method protocol is shorter due to the elimination of the secondary antibody incubation steps.	The indirect method protocol is longer due to secondary antibody incubation and the restinent wash steps.
Multiplexing	Multiplexing is easier with direct detection because multiple primary antibodies from the same host species can be used together.	Indirect method multiplexing is more complicated due to secondary antibody cross-reactivity.
Background	Non-specific background signal is lower in the direct method as secondary antibody mediated cross-reactivity is eliminated.	Non-specific background staining is generally higher in the indirect method due to secondary antibody cross-reactivity.
Flexibility	The direct method is less flexible since the selection of directly conjugated antibodies can be limited.	The indirect method is more flexible because it is possible to use different conjugated secondary antibodies depending on your experiment.

Conjugated Antibodies

Can’t find a labeled antibody? Try searching novusbio.com for your target and the conjugate of interest. Novus Biologicals provides over 55,000 unique antibody-dye combinations. If you do not see a conjugated antibody listed on our website, ask for custom conjugations. In our in-house conjugation facility, Novus’ Lab Team custom labels many of our unconjugated primary antibodies on special request.

Want to label an antibody yourself? Try Novus antibody labeling kits which enable you to conjugate an antibody in as little as 30 seconds! Learn more about antibody labeling kits.