Secondary antibodies for IHC
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When using an indirect detection protocol, selecting the right secondary antibody is essential.
Unlike direct detection using a labeled primary antibody, the use of secondary antibodies enables signal amplification as more than one secondary antibody molecule binds to each primary.
The secondary antibody should be directed against the species in which the primary antibody was raised (ie if a primary antibody raised in rabbit is used, an anti-rabbit secondary antibody raised in a species other than rabbit must be used). It is also important that the isotype that the secondary antibody is raised against matches the primary antibody’s isotype.
Secondary antibodies can be either enzyme-labeled (peroxidase, alkaline phophatase), fluorochrome-labeled (FITC, R-PE, Alexa-Fluor®) or biotinylated.
Our range of biotinylated secondary antibodies for use in ABC (avidin-biotin complex) detection methods can be found here.
Secondary antibodies commonly used for IHC
Secondary antibody | HRP | Biotin |
|---|---|---|
Goat Anti-rabbit (IgG H&L) | ||
Goat Anti-mouse (IgG H&L) | ||
Goat Anti-rat (IgG H&L) | ||
Goat Anti-chicken (IgG H&L) | ||
| See the full range | ||
Generally, affinity-purified antibodies are the most popular as they provide the lowest amount of non-specific binding. However, IgG fractions can also potentially contain very high affinity antibodies and may be useful when an antigen is poorly expressed or in low abundance.
Pre-adsorption of secondary antibodies can reduce non-specific background as they are less likely to show species cross-reactivity or to react with endogenous antigens of the species against which they have been pre-adsorbed. The secondary antibody should be pre-adsorbed against the species in which the sample originated. For example, it is advisable to use a secondary antibody pre-adsorbed against human serum when staining human tissues or cell lines.
More information on pre-adsorbed antibodies can be found here.
F(ab’)2 fragment secondary antibodies
F(ab’)2 fragment secondary antibodies are recommended for staining tissues that are rich in Fc receptors (eg spleen, thymus, blood). F(ab’)2 fragment secondary antibodies, which are smaller and therefore penetrate tissues more easily, are particularly useful for multiple IHC staining.
Secondary antibodies can be either enzyme-labeled (peroxidase, alkaline phophatase), fluorochrome-labeled (FITC, R-PE, Alexa-Fluor®) or biotinylated. Our range of biotinylated secondary antibodies for use in ABC (avidin-biotin complex) detection methods can be found here.
HRP-polymer secondary antibodies for low-expressing proteins
For higher sensitivity, HRP-polymer secondary antibodies use micropolymer technology to form smaller detection complexes that allow better tissue penetration and sensitivity. In addition, HRP-polymer secondaries bind more horseradish peroxidase than standard HRP secondary antibodies, increasing signal.
Host species primary raised | HRP-polymer secondary |
Mouse | |
Rabbit | |
Rat | |
Goat |
View our full range of secondary antibodies here.
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