Vitamin H and Egg White: Streptavidin-Biotin for Immunohistochemistry
If you want to make molecules stick together you need to know about streptavidin/biotin. This article follows on from Mike’s article looking at ‘sandwich’ and ‘amplification’ methods of immunohistochemistry (IHC) and covers how streptavidin-biotin works in IHC, including protocols.
Streptavidin-Biotin
What is it?
Avidin is a natural biotin-binding protein found in egg whites.
Streptavidin is similar to avidin but it synthesised by a number of the Streptomyces family of bacteria and is used to inhibit the growth of competing bacteria.
Biotin is a small molecule B-vitamin (Vitamin B7) also known as Vitamin H (or Coenzyme R). The ‘H’ stands for ‘Haar und Haut’ – German for hair and skin as it contributes to healthy skin, hair and nails.
What Does it Do?
Streptavidin and avidin have an extremely high-affinity for biotin. However, streptavidin is usually favoured over avidin, in that it has a very high specificity for biotin. Steptavidin binding is strong and is via a large number of hydrogen bonds. So strong that this bond is almost as strong as a covalent bond.
Streptavidin/biotin reactions are appealing because of their strong, specific nature. And the fact numerous biotin molecules can be coupled to a single protein/antibody of interest. Thus biotin can be used as a very specific IHC amplification technique.
History of Streptavidin-Biotin in IHC
Streptavidin-biotin and its wonderfully high affinity was first used in research in the 1970’s. In 1979, a team working at the Institut Pastuer published a paper wherein they describe the biotin labelling of antibodies and antigens with biotin. Including demonstrating this techniques usefulness in quantitative immunosassays and IHC (1). Then in 1981, a paper was published on the Avidin-Biotin-Peroxidase Complex (ABC) which is still widely used to date (2). In this paper, the research team developed a new method for use with formalin-fixed paraffin embedded tissue that involves the biotin labelling of secondary antibodies and the subsequent addition of the ABC. Here the biotinlyated secondary antibodies form a link between the primary detection antibody and the avidin/biotin complex.
In 1977, Vector Labs introduced one of the first commercial avidin (or streptavidin)-biotin complexes. They also helped to develop and patent ABC systems and subsequently made these commercially available to the research community. It wasn’t long until other companies started to offer similar commercial products. As well as peroxidase, avidin-streptavidin can be covalently linked to ligands such as fluorescent proteins enabling an even greater range of biological and IHC applications. For example, Molecular Probes (Life Technologies) produce streptavidin conjugated to their Alexa Fluor fluorescent probes offering a range which spans the excitation spectrum from 346 nm to 749 nm.
Know Your Limits
The ABC method has limitations though. Most importantly, endogenous biotin can be found in tissue, especially in the liver, spleen and kidneys. This endogenous biotin expression is highest in frozen tissue sections compared to formalin-fixed-paraffin-embedded tissue. Furthermore, although antigen retrieval techniques are useful for unmasking antigens, retrieval techniques can also increase endogenous biotin availability, causing high background staining. Another problem with using avidin-biotin complexes is that it is a relatively large molecule, which can hinder its penetration into tissue.
Example Protocols
Below are methods to block endogenous biotin, as well as an example streptavidin-biotin IHC method.
Biotin Blocking Protocol
Biotin blocking should be carried out after the normal serum blocking and before incubation with the primary antibody of interest. You can block using this protocol or one of the many commercial kits available.
Materials
- Wash buffer (either PBS or TBS)
- Biotin (make 0.01% in wash buffer)
- Streptavidin or Avidin (make 0.05 % in wash buffer)
Method
- Incubate sections with the streptavidin/avidin solution for 20 minutes. This step will block the endogenous biotin in the tissue
- Wash in wash buffer. This step is important. If missed, then the streptavidin/avidin which is bound to the tissue (step 1) would also bind the biotin-labelled antibody/probe used in the IHC method.
- Incubate sections with the biotin solution for 20 minutes. This step will saturate all of the available biotin binding sites of the free avidin/streptavidin.
- Wash in wash buffer.
- Proceed with application of primary antibody.
Streptavidin-Biotin IHC Protocol
- Dewax and rehydrate slides. See the H&E article for details.
- Perform antigen retrieval if necessary.
- Block with normal serum using the same species as the secondary antibody.
- Carry out the biotin blocking method if necessary.
- Add your primary antibody for the desired time.
- Wash three times in wash buffer.
- Carry out peroxidase blocking. Incubate sections for 20 minutes in a solution of 3% H2O2 in PBS.
- Wash three times in wash buffer.
- Add the biotinylated secondary antibody (in PBS) for at least 30 minutes.
- Wash three times in wash buffer.
- Add an ABC Peroxidase detection solution for 30 minutes (note- these are usually in kit form such as those kits available from Thermo Scientific Pierce and and Vector Labs.
- Wash three times in wash buffer.
- Incubate sections in the peroxidase substrate DAB (3,3’-diaminobenzidine). This usually comes in kit form, such as those available from Abcam or Vector Labs.
- Wash three times in wash buffer.
- Counterstain if necessary.
- Dehydrate through alcohols and into xylene.
- Add coverslip/mounting medium.
And that is how it is done! Hopes that gets you started using streptavidin-biotin in your IHC. Although it can also be used in a wide-variety of other techniques including Western Blots and ELISAs.
References
- Guesdon et al. (1979) The Use of Avidin-Biotin Interaction in Immunoenzymatic Techniques. J Histochem Cytochem 27:1131–9.
- Hsu et al. (1981) Use of Avidin-Biotin-Peroxidase Complex (ABC) in Immunoperoxidase Techniques. J Histochem Cytochem 29:577–580.