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Detection Methods

There are many ways to detect a primary antibody on tissue. Here is a brief guide to the possibilities.

Enzymatic Detection Systems

There are many ways of detecting antibodies in immunohistochemistry, each has its own benefits and drawbacks. This is a rough guide to the possibilities – if in doubt, give us a shout!

Whichever method is used, it is important to run controls to ensure the staining is appropriate. A common negative control is to run the protocol omitting the primary antibody. This is an excellent quick way to ensure that the detection reagents are not causing unwanted staining but should not be confused with validating the primary. (Further information on this can be found in the Vector Webinars)

Detection Methods Key

Method Diagram


Method Protocol Outline



Labelled Primary


  1. Primary antibody
  2. Substrate
  • Fast
  • Background only if primary is binding off target or reporter molecule in tissue
  • Small size of reagent allows good tissue penetration.
  • Low sensitivity
  • No other label choices – new primary conjugate needed
  • Availability of pre-labelled primaries
  • Binding of primary antibody may be affected by conjugation



  1. Primary antibody
  2. Labelled secondary antibody
  3. Substrate
  • Cheaper to change label options- secondaries can be used after different primaries
  • Introduction of cross reactivity issues

Peroxidase, Anti-Peroxidase


Alkaline Phosphatase, Anti-Alkaline Phosphatase



  1. Primary antibody
  2. Secondary antibody
  3. Anti-enzyme, made in same species as primary
  4. Enzyme
  5. (Repeat steps 3&4 if required)
  6. Substrate
  • Can be layered a few times to increase sensitivity
  • Endogenous HRP/AP can cause issues
  • Time consuming compared to methods of similar sensitivity

Labelled Avidin/ Streptavidin



  1. Primary antibody
  2. Biotinylated secondary antibody
  3. Labelled streptavidin
  4. Substrate
  • Small detection label penetrates tissues well.
  • Detection can be ‘mix & match’ for different labels and species
  • Endogenous biotin can cause background

Avidin Biotin Complex ABC


  1. Primary Antibody
  2. Biotinylated Secondary antibody
  3. Avidin Biotin Complex
  4. Substrate
  • Increased number of enzymes per primary compared to LASB method
  • Easy to detect a wide range of species, or choose adsorbed secondary antibodies
  • The ABC layer may be repeated with a biotinylated anti-avidin linker. METHOD
  • Complex needs to be made 30 minutes before use unless a ready to use reagent is bought
  • Larger complexes can have difficulty penetrating tissues, however there are different varieties of ABC kits available. 
  • Endogenous biotin can be an issue
  • Kits available that can detect several species



  1. Primary antibody
  2. Polymer conjugated secondary antibody
  3. Substrate
  • Increased number of enzymes bound to one primary antibody
  • Condensed protocol time compared to ABC method
  • Does not encounter issues that kits using biotin have.
  • Often conveniently provided as Ready-to-Use
  • Required detection reagent for each different species available – ranges may be limited to most popular species of primary antibodies.
  • Ready to use reagents can limit the variables

Amplified Micropolymer Kits


  1. Primary antibody
  2. Secondary antibody
  3. Polymer conjugated anti-secondary
  4. Substrate
  • Very sensitive system in same time as ABC kit
  • Although not as sensitive as tyramide systems, it is a much less expensive option
  • Some attention may need to be given to cross reactivities in the method.
  • Limited range of species detectable with the kits on the market.

Tyramide Signal Amplification



  1. Primary antibody
  2. Biotinylated secondary antibody
  3. ABC/ LSAB
  4. Tyramide
  5. Substrate

* other variations of this method are also possible

  • Tyramide is a relatively small molecule for good tissue penetration
  • Can be used in conjunction with ABC method for best amplification.
  • Biotin version encounters issues with endogenous biotin.
  • HRP conjugated secondaries can be used
  • Pricey
  • Long method

Rolling Circle Amplification


  1. Primer labelled primary
  2. Extend primer with DNA amplification
  3. Detect extended primer with  enzyme labelled probe 
  4. Substrate
  • Not reliant on biotin
  • Using probe technology can reduce chances of off target binding
  • Expensive.
  • Need reagents to  be matched for the technology to work.
  • Time

In general, the more steps or layers there are in a protocol, the more sensitive it is. However, each layer adds time to the method, and can also increase the risk of non-specific staining.

Although some methods appear to offer unlimited amplification, the conditions within the tissue environment can limit the results in practice, and the signal to noise ratio can become problematic.  A more sensitive detection system may be more expensive, but the cost can often be offset in savings on the primary antibody. If the antigen is prevalent, a very sensitive system is not required, however, if searching for the initial mutations in a tumour, more sensitivity may be very desirable. Tailoring the detection system to the experiment should therefore be considered.

For many years clinical labs have found polymer systems to be effective for most samples, and offer a slightly faster turnaround time, enabling automated staining machines to complete another run of slides each day. These polymer systems seem a practical option for many research labs, although those with more varied needs of different species of tissues and antibodies, or tighter grant can value the adaptability of the ABC systems.

The sensitivity of the method also depends on the sensitivity of the substrate used. This will be covered more in the substrate discussion, but if a little more sensitivity in a method is required, investing in a better substrate may offer a simple solution without changing the whole method.

There are further variations to these methods, some labs have used a biotin anti-(strept)avidin after an ABC layer followed by more ABC, some labs have methods that apply a substrate a second time.

Multiple Labelling

Enzymatic substrates can provide a sheltering effect over a label. This can be useful in multiple labelling experiments where the primary antibodies are made in the same species. Unfortunately, the sheltering can also interfere with the subsequent labels binding if the antigens are co-localised. Where antigens are expected in the same cell, and same cell compartment, then fluorescent methods may be required.  

Vector’s IHC Multiplexing guide – for colorimetric multiple labels.

Avidin & Streptavidin – What’s the difference?

Although these 2 molecules often perform the same basic function in assays, there are some differences in these molecules that can have an impact.

Streptavidin is a non-glycosylated 60kDa protein made of 4 sub units, purified from Streptomyces avidinii. Although it has only 30% sequence similarity to avidin, it has nearly identical secondary, tertiary and quaternary structure, and a binding affinity of 1014 M-1 to biotin. However, it does have a sequence that mimics the binding sequence of fibronectin, a universal recognition domain of the extra cellular matrix that specifically promotes cell adhesion. This interaction may cause high background in some applications.

Avidin is a 68kDa basic glycoprotein purified from egg white. With an exceptionally high affinity for biotin (1015 M-1) this bond is essentially irreversible. Different methods of preparation of the avidin can result in avidin displaying modified properties; primarily deglycosylating the avidin can offer a more neutral isoelectric point, and reduces any non-specific interaction with lectins. Other treatments can affect the affinity of avidin – enabling elution of biotinylated reagents under less harsh chemical conditions.

In avidin/biotin or streptavidin/biotin blocking methods, the sections are incubated first with a solution of (strept)avidin to bind to any free biotin in tissues. The free biotin binding sites on the (strept)avidin are then filled with a solution of free biotin – which can only bind to one biotin binding site, effectively preventing further reaction.

It’s good practice to use the correct avidin or streptavidin blocking kit as appropriate, however they can be used fairly interchangeably, bearing in mind that there may be occasions where the glycosylation of avidin, or streptavidin’s mimicry of fibronectin requires the appropriate blocking kit.

It is interesting to note that anti-avidin and anti-streptavidin antibodies exist that do not recognise the other species – so where biotinylated anti-avidin is used to increase a method’s sensitivity, blocking with a streptavidin biotin blocking kit, and vice versa, may be possible.