Best Practice for Secondary Antibody Use in IHC

Using an indirect method of biomolecule detection, such as secondary antibody use, allows for the amplification of signals. This is because more than one secondary antibody can bind to each primary antibody. However, as the method is indirect the correct choice of secondary antibody is important.

The secondary antibody used should be sourced from a species different from the one in which the primary antibody was sourced from. For example, if the primary antibody is sourced from a rabbit, then an anti-rabbit secondary antibody sourced from a different species should be utilized.

Researchers should also make sure that the isotype of the secondary antibody corresponds to the isotype of the primary antibody.

Secondary antibodies can be labeled for detection by a variety of different methods including fluorochrome labeling (e.g. R-PE, FTIC, Alexa-Fluor®), enzyme labeling (e.g. alkaline phosphatase, peroxidase) or biotinylated. Abcam provides a wide range of secondary antibodies with biotinylated labels which can be used in avidin-biotin complex (ABC).

Secondary antibody HRP Biotin
Goat Anti-rabbit (IgGH&L) ab205718 ab207995
Goat Anti-mouse (IgGH&L) ab205719 ab207996
Goat Anti-rat (IgG H&L) ab205720 ab207997
Goat Anti-chicken (IgG H&L) ab205721 ab207998
See the full range

Antibodies purified using affinity chromatography are the most commonly used as they provide the most specific binding. IgG fractions of antibodies can also be used as they can contain antibodies of high affinities which can be helpful for antigens of low abundance or expression.

Non-specific background results can be minimized by pre-adsorbed secondary antibodies. Pre-adsorption results in a reduced propensity of the secondary antibodies to exhibit reactions with endogenous antigens of the species they have been pre-adsorbed against, and a lower species cross-reactivity.

For example, when staining tissues or cells of human origin, it is best practice to use a secondary antibody which has been pre-adsorbed against human serum.

F(ab’)2 Fragment Secondary Antibodies

When staining tissues which have a high Fc receptor content, such as the thymus gland, blood or the spleen, the use of F(ab’)2 fragment secondary antibodies is recommended. F(ab’)2 fragment secondary antibodies are also useful tools for multiple IHC staining as there fragmented nature means they are smaller, allowing them to enter tissues more readily.

As with conventional secondary antibodies, F(ab’)2 fragment secondary antibodies can be labeled for detection by a variety of different methods including fluorochrome labeling (e.g. R-PE, FTIC, Alexa-Fluor®), enzyme labeling (e.g. alkaline phosphatase, peroxidase) or biotinylated.

Abcam provide a wide range of secondary antibodies with biotinylated labels which can be used in avidin-biotin complex (ABC).

Detecting Low Protein Concentrations with HRP-Polymer Secondary Antibodies

For the detection of proteins that are only expressed in small volumes secondary antibodies of a greater sensitivity, such as HRP-polymer secondary antibodies, must be used. HRP-polymer secondary antibodies exhibit better sensitivity and tissue penetration by the use of micro polymer technology.

The signal of experiments involving HRP-polymer secondary antibodies is also increased as the secondary antibodies bind more to peroxidase than conventional HRP secondary antibodies.

Host species primary raised HRP-polymer secondary
Mouse Goat Anti-Mouse IgG H&L (HRP polymer) (ab214879)
Rabbit Goat Anti-Rabbit IgG H&L (HRP polymer) (ab214880)
Rat Goat Anti-Rat IgG H&L (HRP polymer) (ab214882)
Goat Donkey Anti-Goat IgG H&L (HRP polymer) (ab214881)

View our full range of secondary antibodies here.

About Abcam

Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.

Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.


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Last updated: Feb 13, 2019 at 12:36 PM

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