Visualization systems for lectin histochemistry and immunohistochemistry

Visualization – since the binding of the antigen to the antibody (in the case of immunohistochemistry) or the oligosaccharide residue to the lectin (in the case of lectin histochemistry) is not visible, it must be visualized in the next step. The visualization methods are similar for both types of bonds.

Visualization methods


 * 1) Formation of a colored compound in situ by enzyme histochemistry
 * 2) Fluorochrome
 * 3) Avidin-biotin
 * 4) Metal
 * 5) Digoxygen

Formation of a colored compound in situ by enzyme histochemistry
The antibody is labeled with an enzyme. The enzyme breaks down the substrate we supplied. The product then reacts with the chromogen to form a colored compound.

The most frequently used enzymes, their substrates and chromogens:

Alkaline Phosphatase (ALP)
We must maintain an alkaline environment (pH 9).

Substrate :
 * glycerphosphate (obsolete)
 * α-naphthyl phosphate (NP)
 * bromo-chloro-indolyl phosphate (BCIP)

Chromogen:
 * nitrotetrazolium blue (NBT) – blue reaction
 * fast red – red reaction – soluble in alcohol and xylene

Horseradish peroxidase (HRP)
Substrate :
 * Hydrogen peroxide

Chromogen:
 * Diaminobenzidine (DAB) - black-brown reaction - insoluble, osmiophilic
 * Aminoethylcarbazole (AEC) - red reaction - the resulting product is soluble in alcohol and xylene, so we must use water-soluble media such as gelatin.

Fluorochrome
It is a fluorescent dye. These substances have the ability to receive energy in the form of excitation light. Excitation light must have a short wavelength and therefore have high energy. Thanks to the received energy, the electrons of the fluorochrome jump to a higher energy level, where, however, they cannot stay, and so they return to their original energy level. When it jumps back, it emits energy in the form of fluorescence radiation, which has a lower energy and thus a longer wavelength. We are able to observe fluorescent light with a fluorescence microscope.

The most common types of fluorochromes:


 * 1) FITC (fluorescein isothiocyanate) green
 * 2) TRITC (rhodamine) red
 * 3) Dapi (diamidinophenylindole) blue

Avidin - biotin
Avidin is an egg glycoprotein. Biotin is a vitamin H belonging to the group of B vitamins (some literature mentions vitamin B7). 1 molecule of avidin can bind up to 4 molecules of biotin. It is a very strong non-immunogenic bond. This can be used for signal amplification. It is used, for example, if the structure we are looking for is represented to a small extent. The avidin molecule is not fully occupied by biotin, to which peroxidase (or alkaline phosphatase) is bound. The free space is for the biotinylated antibody.

Metal
Metal labeling of antibodies is used for electron microscopy methods. Heavy metals are used for marking. They must be electron dense.

The most commonly used metal: colloidal gold particles

Digoxigenin
Digoxigenin is obtained from foxglove. Used in lectin histochemistry. The lectin is labeled with digoxigenin and then an anti-digoxigenin antibody is subsequently used. The antibody is labeled with a fluorochrome or an enzyme.

Direct method
It is the simplest way of locating the antigen (or oligosaccharide residue) in the tissue. We only use a primary antibody that is already directly labeled with a fluorochrome, enzyme or metal. This method is no longer widely used, as the demonstrated structure must be sufficiently represented in the tissue and must be well accessible to the antibody. It is not so sensitive on paraffin sections.

Indirect two-step method
In this method, we use a primary antibody directed against the desired antigen (in the case of lectin histochemistry, the primary antibody is directed against the lectin), which is not labeled. We attach a secondary antibody to the primary antibody, which binds to the Fc fragment of the immunoglobulin of the animal from which the primary antibody was obtained. The secondary antibody is already labeled with a fluorochrome, enzyme or metal.

Indirect three-step method
It is an amplification method used to amplify a signal. The signal needs to be amplified, for example: if there is a low amount of antigen molecules in the tissue.


 * 1) stage- We will use a primary antibody against the antigen of interest. It is not marked.
 * 2) stage – an antibody is added that is directed against the immunoglobulins of the animal from which the antibody is primary and the antibody is used in the 3rd stage. This antibody is also unlabeled. It is referred to as a binding antibody.
 * 3) stage– a labeled complex is applied, e.g.:

PAP – peroxidase-anti-peroxidase complex APAAP – alkaline phosphatase-anti-alkaline phosphatase (in preparations where endogenous peroxidase activity is high and we cannot be sure of its full inactivation)

A three-step method using ABC or SABC
ABC-avidin-biotin complex

SABC-streptavidin-biotin complex


 * 1) stage- binding of the primary antibody (unlabeled) to the antigen of interest.
 * 2) stage– binding of the biotinylated secondary antibody to the primary antibody. She will create a bridge.
 * 3) stage– establishing ABC or Bridged SABC (biotinylated antibody). Biotin in the complex is labeled with peroxidase or alkaline phosphatase, and their activity indicates the places where the desired structure is located and also to what extent. Visualization is performed using enzyme histochemistry.