Information AboutImmunohistochemical Staining |
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In the procedure depending on the purpose and the thickness of the experiment, either thin (about 20 μm ) slices are taken of the tissue of interest or if the tissue is not very thick and penetrable it is used as a whole. The slicing is usually accomplished through the use of a cryostat, and slices are mounted on slides. "Free-floating IHC" uses slices that are not mounted, normally produced using a vibrating microtome. The protein to be detected or estimated is called the antigen and the protein which is used to detect it, ie. which can bind to the Antigen is called the Antibody . In both cases, tissue is treated to rupture Membrane s usually by using a kind of detergent called Triton . Antibodies to the protein of interest are then applied, and can now penetrate the tissue. After Incubation with " Primary " antibody, the tissue is incubated with a " Secondary " antibody directed at the primary antibody. This secondary antibody is typically conjugated to either Biotin or a reporter Enzyme such as Alkaline Phosphatase or Horseradish Peroxidase . In the most common procedure, a biotinylated secondary antibody is coupled with Streptavidin-horseradish Peroxidase . This is reacted with 3,3'-Diaminobenzidine (DAB) to produce a brown staining wherever primary and secondary antibodies are attached in a process known as DAB staining. The reaction can be enhanced using Nickel , producing a deep purple/grey staining. Secondary antibodies Conjugated to Fluorescent agents, such as the Alexa Fluoro family, are also frequently used for detection of proteins in IHC procedures. Protein concentration is generally measured by densitometry analysis, where the intensity of staining correlates with the amount of the protein of interest. IHC is an excellent detection technique and has the tremendous advantage of being able to show exactly where a given protein is located within the tissue examined. This has made it a widely-used technique in the Neurosciences , enabling researchers to examine protein expression within specific brain structures. Its major disadvantage is that, unlike immunoblotting techniques where staining is checked against a molecular weight ladder, it is impossible to show in IHC that the staining corresponds with the protein of interest. For this reason, primary antibodies must be well-validated in a Western Blot or similar procedure. The technique is even more widely used in diagnostic Surgical Pathology for typing tumors (e.g. carcinoma vs melanoma). |
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