The definitive guide to HRP secondary antibodies for immunohistochemistry (IHC)

Introduction

Immunohistochemistry (IHC) is the experimental process whereby primary antibodies are used to detect proteins (antigens) in cells within a tissue section.  For indirect detection, generally a HRP conjugated secondary antibody against the primary antibody is used.  Staining is visualized using substrates such as DAB.

To help scientists discover more, Abcam has developed a portfolio of over 300 HRP secondary antibodies that have been validated in assays such as western blot, ELISA and immunohistochemistry.  Advantages of the range include:

FeatureBenefit
High sensitivityDetect lowly expressed targets with high signal-to-noise ratios due to the optimal number of HRP molecules per antibody
Large dilution range from 1:2000 to 1:20,000Optimal performance with minimal background
Large range to select your perfect HRP secondary antibody

Host species - goat, rabbit, mouse, donkey, sheep, rat, chicken and alpaca.

Target species - rabbit, mouse, donkey, human, duck, cow, pig and monkey.

Pre-adsorbedIdeal for single, double or tripe IHC labeling experiments. Pre-adsorbed HRP secondary antibodies minimize cross species reactivity.
Highly validatedGuaranteed performance in the assay of choice and covered by Abcam's Abpromise.


Popular products within Abcam's HRP secondary antibody portfolio

HRP Secondary AntibodyNot pre-adsorbedPre-adsorbed
Anti-rabbit HRP (IgG H&L)

ab205718

ab97080
Anti-rabbit ​HRP  (Fc)​ab97200ab98467
Anti-mouse HRP (IgG H&L)

ab205719

ab97040
Anti-mouse HRP (IgG Fcab97265ab98717
Anti-goat HRP (IgG H&L)ab205723ab7125
Anti-rat HRP IgG H&L)

ab205720

ab7097
Anti-chicken HRP (IgG H&L)

ab205721

No

Anti-mouse isotype specific HRP secondary antibodies ideal for immunohistochemistry double or triple staining

The issue - the majority of primary antibodies have been generated in either rabbit or mice, therefore for a researcher to perform a double IHC staining experiment, primary antibodies from two different species must be used, especially when using secondary antibodies. However, scientists will often encounter situations where antibodies against the two target proteins have been generated in the same species, for example two mouse monoclonal primary antibodies.

The solution - all is not lost if the antibody isotypes are different (IgM and IgG2a). An experiment may be designed whereby two anti-mouse secondary antibodies (one conjugated to HRP, the other to Alkaline Phosphatase (AP)) may be used. Each targeting the different antibody isotypes (ie IgG2a and IgM).

We currently offer several anti-mouse isotype specific antibodies conjugated to HRP or AP - a great range for double or triple labeling.

Anti-mouse isotype specific HRP secondary antibodies

HRP secondary antibodyAbIDPre-adsorbed
Anti-mouse HRP (IgM)ab97230No
Anti-mouse HRP (IgG2a H)ab97245No
Anti-mouse HRP (IgG1 H)ab97240No
Anti-mouse HRP (Kappa L)ab99632No


Anti-mouse isotype specific AP secondary antibodies

AP secondary antibodyAbIDPre-adsorbed
Anti-mouse AP (Fc)ab98710Yes
Anti-mouse AP (IgG2b H)ab98700Yes
Anti-mouse AP (Kappa L)ab99631No


Handy tips for optimizing your IHC experiment when using a HRP secondary antibody

  • Choose the right HRP secondary antibody

It is important to make sure your HRP secondary antibody is compatible with your primary. For instance, if the primary has been raised in rabbit, you should use an anti-rabbit HRP secondary.  Additional tips are discussed in our 'Secondary antibody selection guide'.

  • What starting dilution should I use for a HRP seconday antibody during IHC staining?

The optimal dilution for a HRP secondary antibody will change for each individual primary antibody. We suggest a pilot titration to be performed over a large range from 1 in 1,000 to 1 in 100,000.

  • What buffer do you suggest for a HRP labeled secondary antibody?

Use 1% BSA with TBS.  Abcam recommends TBS as it has been shown to give a cleaner background than PBS.  If high background staining is still encountered you may wish to increase the percentage of BSA or use 5% milk in TBS.

Please note, the secondary antibody may cross react with endogeneous immunoglobulins in the tissue.  This is minimized by pre-treating the tissue with normal serum from the species in which the secondary was raised.  The use of normal serum before the application of the primary also eliminates Fc receptor binding of both the primary and secondary antibodies.  BSA is included to reduce non-specific binding caused by hyrdophobic interactions.

  • Do I need a pre-adsorbed secondary antibody?

Pre-adsorbed 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 HRP secondary antibody pre-adsorbed against human serum when staining human tissues or cells.  

  • What is a good control to demonstrate specific staining of the secondary antibody?

To demonstrate that the secondary antibody has reacted specifically with the primary, we recommend a control test which includes a HRP secondary antibody wit the primary antibody omitted.  If staining is observed in the control sample the results suggest the HRP secondary antibody is reacting non-specifically with endogenous immunoglobulins on the tissues.

  • Incubation time

One hour at room temperature should be sufficient.  If the signal is weak, increase incubation time to overnight at four degree Celsius.

Get HRP secondaries for my experiments.


Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.


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