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Immunohistochemistry Basis(Continued) – Tissue Preparation

Abstract:      In the previous article, we have preliminarily learned about immunohistochemistry. Then, antibody preparation involves the immunization of purified antigens (e.g. mouse, rabbit, goat, horse etc).
Keywords:   Immunohistochemistry, Tissue Preparation, Antibody Preparation

1. Immunohistochemistry Antibodies

Various animals can be applied to produce polyclonal antibodies, especially rabbit, horse, goat and chicken etc. Polyclonal antibodies have a high affinity and wide reactivities. However, compared with monoclonal antibodies, the specificity is lower. Because polyclonal antibodies can bind with multiple antigenic epitopess in fixed tissues, the sensitivity is improved. Usually, antibodies which can recognize strong immunogenicity and weak antigenic epitopes exist in polyclonal antibodies reagents. Hence, polyclonal antibodies may be very inconsistent. The binding of multiple epitopes is a advantage.(It can bind with several antigenic epitopes in the same protein. Antigens can be more likely to be detected.) The possibility of cross reaction with other proteins which have similar antigenic epitopes will be increased, resulting in the false positive.

Immunohistochemistry Result_1

Monoclonal antibodies are usually produced by mice. The technology is developed by Kohler and Milstein. Compared with polyclonal antibodies, the advantage of monoclonal antibodies is high specificity which decreases the cross reaction with other antigen(It's impossible to completely remove the reaction). If the cross reaction appears, the possible reason is that antigenic epitopes targeted by monoclonal antibodies just consist of several amino acids. However, the antigenic epitope exists in various proteins and polypeptides. Sometimes, it's very hard to confirm the non-specific immune response is caused by common antigenic epitopes(cross reaction) or protein crosslinks when the tissue is fixed by aldehyde fixatives. In some situations, the background colour of monoclonal antibodies caused by non-specific immunoglobulins can decrease ascites or disappear in the culture supernatant of hybridoma cells.

2. Tissue Preparation for Immunohistochemistry

The tissue preparation determines whether the immunohistochemical reaction can succeeds to a great extent, which mainly depends on antigen types to be tested in some ways. E.g. During the fixation of formaldehyde, some antigens will be destroyed. Hence, these tissues should be frozen or fixed by other stationary liquids. A principle for testing antigens should be followed: samples are required to be collected and treated before the decomposition of tissues.

Immunohistochemistry Result_2

3. Tissue Fixation for Immunohistochemistry

Tissues which are not instantly frozen should be fixed at once. Tissue fixation is very necessary. Reasons are as follows(functions of tissue fixation):

  • Preserve cell components like soluble protein and structural protein etc;
  • Prevent the degradation and translocation of cell components like antigen and some enzymes;
  • Stabilize cell components to reduce harmful effects of next steps;
  • Help traditional immunohistochemistry staining and immunostaining. Two types of fixatives are mainly applied in histopathology: crosslinking fixatives(non-coagulant) and coagulant fixatives.

Formaldehyde is the golden fixative in regular histochemistry and immunohistochemistry. It can preserve most polypeptide and general structure of organelle, and can also react with nucleic acid. However, carbohydrates are not influenced. When the calcium ion is contained, it's the good protective reagent for lipid. The basic fixing mechanism of formaldehyde: formalin and active amido without charges (-NH or -NH2) form a addition product. Finally, modifications of the methylene bridge (crosslinking) are formed in the tertiary and quaternary structure of proteins. Formaldehyde fixation is a gradual process with dependent time and temperature. Over fixation will result in the immunohistochemical false negative outcome caused by over crosslinking. However, insufficient fixation will generate unexpected results. There is no the most suitable fixing time for the fixation of each antigen.

Immunohistochemistry Result_3

Many substitutes of formalin are some coagulant fixatives, which can destroy hydrogen bond to precipitate proteins instead of protein crosslinking. The most typical non-crosslinking fixative is ethanol. Other fixatives applied in immunohistochemistry are glyoxal(dialdehyde), the mixture of glyoxal and ethanol, 4% paraformaldehyde and zinc formalin etc. Compared with formalin, their effects on protein conformational changes are not obvious. It's suggested to follow instructions recommended by the antibody manufacturer to find the most suitable fixing method. For immunohistochemical primary antibodies, if staining results for frozen section are obtained, the staining of fixing tissue is pending for testing. It's suitable to use 4% paraformaldehyde or zinc formalin for fixing paraffin-embedded tissues during testing the staining of fixing tissue. This method is good for using paraffin-embedded tissues. Because different fixatives variably influence immune response, the tissue paraffin fixed by non formaldehyde fixative should be properly labelled before archiving.

4. FineTest IHC Antibodies

FineTest offers IHC antibodies for research use. Browse a list of FineTest antibody.