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Introduction to Antibodies - Immunohistochemistry
Immunohistochemistry / Immunocytochemistry Procedures
Immunohistochemistry / Immunocytochemistry Procedures
In these techniques an antibody is used to link a cellular antigen specifically to a stain that can be more readily seen with a microscope. Detection of antigens in tissues is known as immunohistochemistry, while detection in cultured cells is generally termed immunocytochemistry. For both, there is a wide range of specimen source, antigen availability, antigen antibody affinity, antibody type, and detection enhancement methods.
Thus optimal conditions for immunohistochemical or immunocytochemical detection must be determined for each individual situation, dependent on the above variables. As for all procedures, reference should be made to individual product data sheets and published literature. Also, the Internet today contains a tremendous amount of useful information on Immunohistochemistry/ immunocytochemistry. Quick searches through google.com or similar search engines are highly recommended
Specimen Preparation:
Fixatives
Section Type
Fixatives:
Fixatives are needed to preserve cells and tissues in a reproducible and life-like manner. To achieve this, tissue blocks, tissue sections, cell growths or smears are either immersed in a fixative fluid, or in cases where whole animal systems are studied, the animal is perfused with fixative via its circulatory system (For perfusion protocol, see www.chemicon.com/techsupp/Protocol/perfusion.asp). In the case of cells in culture, cell preparations are either submerged or simply air-dried. Fixatives stabilize cells and tissues thereby protecting them from the rigors of subsequent processing and staining techniques. For immunological studies, fixation is especially imperative to ensure the adequacy of the specimen and target antigens. Tissues have differing protein content and structural arrangement, thus they have a variable ability to retain their structure without significant fixation. Incorrect specimen preparation can block or impede antigen labeling in tissue and cells. Unfortunately, the methods that are best for the preservation of tissue structure do so by altering proteins, thereby masking some epitopes, and sometimes preventing the detection of the desired target protein. In cases of failure it is important to experiment with multiple different fixatives and antigen retrieval methods prior to “giving up” on a specific stain.
Fixatives may work by several means: formation of crosslinkages (e.g., aldehydes such as glutaraldehyde or formalin); protein denaturation by coagulation (e.g., acetone and methanol); or a combination of the above. Fixation strengths and times must be optimized so that antigens and cellular structures can be retained and epitope masking is minimal. Requirements for fixation can vary widely between tissues. For example when using antibodies to probe for neurotransmitter substances, most tissues must be either immersion fixed with a mixture of glutaraldehyde and paraformaldehyde, or with paraformaldehyde alone. Both acetone and methanol (precooled to -20°C) have been used successfully as fixatives for frozen tissue in other instances.
Immunocytochemistry
Fixation of cultured cells for immunocytochemistry also requires careful consideration; in general, fixation strengths and times are considerably shorter for cells than on the thicker, structurally complex tissue sections. For immunocytochemistry, sample preparation essentially entails fixing the target cells to the slide. Depending upon the needs of the experiment, cells can be simply harvested and “dropped” on to a slide, fixed and air-dried, as is often done with clinical or diagnostic studies where speed and a simple “yes/no” answer is all that is needed. Alternatively, where information on the structural location within the cell is needed, cells are often cultured directly on prepared slides or coverslip material, which are then simply washed and fixed in place prior to staining.Consulting published literature relating to the tissue/proteins of interest is well worth the time invested. See
The soures
http://www.chemicon.com/resource/ANT...menpreparation
http://www.sigmaaldrich.com/Area_of_...istry.html#1d1
Thus optimal conditions for immunohistochemical or immunocytochemical detection must be determined for each individual situation, dependent on the above variables. As for all procedures, reference should be made to individual product data sheets and published literature. Also, the Internet today contains a tremendous amount of useful information on Immunohistochemistry/ immunocytochemistry. Quick searches through google.com or similar search engines are highly recommended
Specimen Preparation:
Fixatives
Section Type
Fixatives:
Fixatives are needed to preserve cells and tissues in a reproducible and life-like manner. To achieve this, tissue blocks, tissue sections, cell growths or smears are either immersed in a fixative fluid, or in cases where whole animal systems are studied, the animal is perfused with fixative via its circulatory system (For perfusion protocol, see www.chemicon.com/techsupp/Protocol/perfusion.asp). In the case of cells in culture, cell preparations are either submerged or simply air-dried. Fixatives stabilize cells and tissues thereby protecting them from the rigors of subsequent processing and staining techniques. For immunological studies, fixation is especially imperative to ensure the adequacy of the specimen and target antigens. Tissues have differing protein content and structural arrangement, thus they have a variable ability to retain their structure without significant fixation. Incorrect specimen preparation can block or impede antigen labeling in tissue and cells. Unfortunately, the methods that are best for the preservation of tissue structure do so by altering proteins, thereby masking some epitopes, and sometimes preventing the detection of the desired target protein. In cases of failure it is important to experiment with multiple different fixatives and antigen retrieval methods prior to “giving up” on a specific stain.
Fixatives may work by several means: formation of crosslinkages (e.g., aldehydes such as glutaraldehyde or formalin); protein denaturation by coagulation (e.g., acetone and methanol); or a combination of the above. Fixation strengths and times must be optimized so that antigens and cellular structures can be retained and epitope masking is minimal. Requirements for fixation can vary widely between tissues. For example when using antibodies to probe for neurotransmitter substances, most tissues must be either immersion fixed with a mixture of glutaraldehyde and paraformaldehyde, or with paraformaldehyde alone. Both acetone and methanol (precooled to -20°C) have been used successfully as fixatives for frozen tissue in other instances.
Immunocytochemistry
Fixation of cultured cells for immunocytochemistry also requires careful consideration; in general, fixation strengths and times are considerably shorter for cells than on the thicker, structurally complex tissue sections. For immunocytochemistry, sample preparation essentially entails fixing the target cells to the slide. Depending upon the needs of the experiment, cells can be simply harvested and “dropped” on to a slide, fixed and air-dried, as is often done with clinical or diagnostic studies where speed and a simple “yes/no” answer is all that is needed. Alternatively, where information on the structural location within the cell is needed, cells are often cultured directly on prepared slides or coverslip material, which are then simply washed and fixed in place prior to staining.Consulting published literature relating to the tissue/proteins of interest is well worth the time invested. See
The soures
http://www.chemicon.com/resource/ANT...menpreparation
http://www.sigmaaldrich.com/Area_of_...istry.html#1d1
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