Which reagent is best for IHC?
Immunohistochemistry (IHC) is one of the most commonly used techniques involving antibodies and CiteAb now lists over half a million antibodies for IHC.
We have previously written blogs providing an introduction to IHC and also our thoughts on whether to use polyclonal or monoclonal antibodies for IHC. Here we look at what chromogenic reagents you should use to develop your IHC reactions.
To do this we scoured the papers that use IHC to see which reagents you seem to prefer. We found three options that were commonly used;
3,3′-diaminobenzidine (DAB)/H2O2
Many papers we looked at used a secondary antibody coupled to Horseradish Peroxidase (HRP) and then DAB combined with H2O2 as a substrate for the chromogenic reaction. This is perhaps one of the simplest methods to carry out a chromogenic reaction for IHC [1]. DAB can be purchased from a range of vendors and often comes ready diluted or in easy to use tablet form. The DAB is also commonly sold with the H2O2 that is required for the colour reaction.
ABC Systems
These systems makes use of a biotinylated antibody (normally a secondary antibody) and then addition of an avidin/biotinylated enzyme complex [2]. The Avidin has a very high affinity for biotin and also four biotin binding sites, allowing large complexes to form which give the system great sensitivity. A number of enzymes can be used, with HRP being particularly common. ABC kits are available from many vendors, with the one from Vector Laboratories being particularly frequently used.
Polymer Systems
These approaches make use of a polymer which is labelled with an appropriate enzyme and coupled to a secondary antibody [3]. A substrate, such as DAB for a HRP coupled polymer, is then added to allow the colour reaction to occur. These systems have the advantage of not containing biotin or avidin. This gets round potential issues with background staining, which can occur due to the presence of endogenous biotin in certain tissues. These systems are supplied by a range of companies, including by EnVision (DAKO), Ultravision (Thermo), OmniMap (Ventana) and NovaLink (Leica).
Which is best?
But which is best? Is a simple DAB/H2O2 system suitable? Is a polymer IHC system better than an ABC system? We struggled to find published validation which answers these questions, but one study did compare different polymer systems [4]. However, the common use of these methods shows they can all produce good data and it may depend on the specific combination of antibody and tissue as to which is best for your experiment.
Hopefully, we have provided a starting point to help you get started with your experiments and we wish you good luck with your IHC!
If you have any comments please add them below or tweet us at @CiteAb.
Andy and the CiteAb team
Image Credits
Front page image: Ben Sharpe (University of Bath, UK)
Main image: Ben Sharpe (University of Bath, UK)
References
1 – Ramos-Vara, J. A (2011). Principles and methods of immunohistochemistry. Drug Safety Evaluation Methods in Molecular Biology. Volume 691, 2011, pp 83-96, 201
http://link.springer.com/protocol/10.1007/978-1-60761-849-2_5
2 – Vector Stain ABC systems. Vector Laboratories Website. https://www.vectorlabs.com/uk/catalog.aspx?catID=42
3 – Immunohistochemistry Staining Methods, 5th Edition. Published by DAKO. http://www.dako.com/08002_ihc_staining_methods_5ed.pdf
4 – Skaland I, Nordhus M, Gudlaugsson E, Klos J, Kjellevold KH, Janssen EA, Baak JP. Evaluation of 5 different labeled polymer immunohistochemical detection systems. Appl Immunohistochem Mol Morphol. 2010 Jan;18(1):90-6. doi: 10.1097/PAI.0b013e3181b0eaad.
PMID: 19661787
