There are many critical steps in performing IHC. These include proper handling of the specimen, appropriate fixation, paraffin block preparation, antigen retrieval, selection and preparation of antibody and reagents, incubation, washing, and counterstaining [2].
Table of Contents
What are the steps of IHC?
There are many critical steps in performing IHC. These include proper handling of the specimen, appropriate fixation, paraffin block preparation, antigen retrieval, selection and preparation of antibody and reagents, incubation, washing, and counterstaining [2].
How is IHC staining done?
Immunohistochemical staining is accomplished with antibodies that recognize the target antigen. Since antibodies are highly specific, the antibody will bind only to the antigen of interest in the tissue section. The antibody-antigen interaction is then visualized using different detection systems.
What are the different detection methods for IHC?
Various Detection Methods for IHC. Choosing the Right Reporter Enzyme and Chromogen. Multiplexing Chromogenic IHC. Common Counterstains for IHC.
What is the first step in immunohistochemistry?
The first stage of IHC is the application of a primary antibody that binds specifically to the target antigen. There are two main types of antibody, polyclonal and monoclonal.
Why we use dab in IHC?
DAB staining is also heat-resistant, so it can be used in double labeling IHC/ISH experiments, and is extremely stable – in fact stained slides are often stable for many years. When used together with a nickel or cobalt solution as a DAB enhancer, DAB staining becomes a more intense, black color.
What is immunocytochemical staining?
Listen to pronunciation. (IH-myoo-noh-SY-toh-KEH-mih-stree) A laboratory method that uses antibodies to check for certain antigens (markers) in a sample of cells. The antibodies are usually linked to an enzyme or a fluorescent dye.
What is ICC staining?
ICC refers to the staining of isolated or cultured intact cells where samples may be from tissue culture cell lines, either adherent or in suspension. Prior to staining, IHC and ICC samples are also processed differently.
What is DAB staining?
In DAB staining, DAB is oxidized by hydrogen peroxide in a reaction typically catalyzed by horseradish peroxidase (HRP). The oxidized DAB forms a brown precipitate, at the location of the HRP, which can be visualized using light microscopy.
What is HRP staining?
Horseradish peroxidase (HRP) is a 44-kDa protein that catalyzes the oxidation of substrates in the presence of hydrogen peroxide, resulting in a colored product or the release of light as one product of the reaction (chemiluminescence).
What color is DAB staining?
brown
In DAB staining, DAB is oxidized by hydrogen peroxide in a reaction typically catalyzed by horseradish peroxidase (HRP). The oxidized DAB forms a brown precipitate, at the location of the HRP, which can be visualized using light microscopy.
What is peroxidase staining?
Peroxidase stain. (A) Large promyelocyte stains intensely (blue-black color) because of the abundant primary granules in cytoplasm (asterisk). Myelocytes and segmented neutrophil also stain but less intensely because of the decreasing concentration of primary granules.
What is ICC immunocytochemistry ICC and immunohistochemistry IHC?
Immunohistochemistry (IHC) and immunocytochemistry (ICC) are techniques employed to localize antigen expression and are dependent on specific epitope-antibody interactions. IHC refers to the use of tissue sections, whereas ICC describes the use of cultured cells or cell suspensions.
What is DAPI staining used for?
DAPI staining was used to determine the number of nuclei and to assess gross cell morphology. Following light microscopic analyses, the stained cells were processed for electron microscopy. Cells stained with DAPI showed no ultrastructural changes compared to the appearance of cells not stained with DAPI.
What is chromogen in IHC?
Chromogens are chemical compounds used in IHC labeling procedures that produce a colored product that can be visualized by light microscopy. The choice of chromogen to use is directly related to the enzyme used in the test.
What is DAB used for in IHC?
DAB (3, 3′-diaminobenzidine) produces a dark brown reaction product and can be used for both immunohistochemical and blotting applications. DAB is effective as a single label or as a second color for multiple antigen labeling. Because of its heat-resistance, DAB can be used in IHC/ ISH double labeling applications.
Why is HRP used in IHC?
Antibody–HRP conjugates are superior to antibody–AP conjugates with respect to the specific activities of both the enzyme and antibody. In addition, its high turnover rate, good stability, low cost and wide availability of substrates makes HRP the enzyme of choice for most IHC applications.
Why we use DAB in IHC?
Why is DAB used in IHC?
What is PAP in immunohistochemistry?
The peroxidase, anti-peroxidase (PAP) method of IHC staining is an indirect staining method and was the first effective method of applying antibodies to paraffin-embedded tissue sections when initially developed. This method has a high degree of sensitivity, as well as specificity and stability.