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Immunohistochemistry methods

Methods of immunohistochemistry

In our laboratory we have firmly established a wide range of immunohistochemical stainings and special detections. The markings are usually made with durable colour markers such as DAB or ImmunoGold. We also carry out fluorescent markings on request, as well as double and triple markings.

We establish detection reactions with classical colour substrates (e.g. DAB) as well as with modern fluorescent dyes of your choice. We have many years of expertise in IHC on human, zoological and botanical tissues with numerous mono- and polyclonal antibodies.

Due to the reaction of the primary antibody with the desired antigen, the immune detection is not yet visible. In the following, different methods for the visualization of the antigen-antibody complex will be presented:

Direct method

With the so-called direct method, the primary antibody is bound directly to a fluorescent dye or to an enzyme which later converts a dye. With this method, the background is usually relatively small, since no further secondary antibodies or other substances involved in indirect methods can bind to the tissue. However, with the direct method, there is no signal amplification.

Indirect methods

In both cases, the Fc part of the primary antibody is bound by a secondary antibody directed against the species from which the primary antibody was derived.

In the two-step method, a fluorochrome or gold particle is bound to this secondary antibody, which can be directly visualized. often the secondary antibody is also conjugated with an enzyme (e.g. peroxidase), with which a substrate can then be converted.

In the three-step method, another tertiary antibody is bound to the enzyme-coupled secondary antibody using the same enzyme. With the indirect method, the visualization is achieved by the secondary antibody and its conjugate. The visualization can be performed directly, i.e. without further steps (e.g. fluorescence), or a color substrate, e.g. 3,3'-diaminobenzidine tetrahydrochloride (DAB), is converted in several steps.

The indirect method provides a small (two-step) to strong signal amplification (three-step), since among other things several secondary antibodies can bind to a primary antibody and more fluorochromes, gold particles or enzymes are accumulated. However, the indirect method also has more background than the direct method, since the secondary antibody can also bind to tissue unspecifically. This is reduced by the use of corresponding normal sera.

PAP, APAAP - Method

The PAP method is based on an antibody-enzyme complex consisting of peroxidase-antiperoxidase and TWO antibodies, whereas the APAAP method is based on an antibody-enzyme complex of alkaline phosphatase-anti-alkaline phosphatase with only ONE antibody, in both systems the primary antibody is bound to its Fc part with only one antibody binding site of the secondary antibody, which can also be well understood as a bridge antibody.To ensure that only one antigen binding site of the bridge antibody binds the Fc part of the primary antibody and one antigen binding site remains free, an excess of secondary antibody is used to bind the Fc part of a third antibody, which is part of the PAP or APAAP complex, to the still free second binding site of the secondary antibody.The antibodies of the complex must originate from the same species as the primary antibody used, because otherwise no binding with the secondary antibody will occur.

(Strept) avidin biotin methods

Biotin and avidin have a very high affinity to each other, which is used in different immunohistochemical methods.In all these methods a biotin conjugated secondary antibody is used.Avidin or streptavidin can now bind to the biotin of the antibody.different methods can now be used for visualization.a fluorochrome can be bound to the avidin/streptavidin, which in contrast to a fluorochrome bound directly to the secondary antibody, leads to a signal amplification.Avidin can also be linked to an enzyme (labelled avidin biotin technique, LAB), which later converts into a colour substrate. Alternatively to a fluorochrome or a single enzyme, a complex of avidin, biotin and enzyme can also be linked to avidin.This combination of avidin and biotin with an enzyme is then called the ABC complex. Here, too, the bound enzyme is able to convert dye. The ABC method offers a strong signal amplification because the relatively large complex can carry more enzyme particles than the other methods.

Use of polymers

Another method to obtain an antibody signal is the use of polymers. On average, they can bind ten antibodies and about 70 enzyme molecules. Polymers are thus suitable for decisively amplifying the antibody signal. The resulting increased sensitivity is due to a reduction in unspecific binding. The background signal is therefore substantially weak and this method allows a double staining of two different antigens simultaneously.