Immunohistochemical procedure

Storage and preparation of polyclonal and monoclonal antibodies are very important for successful immunostaining. When a new antibody arrives in a laboratory, special attention should be paid to its storage instructions and optimal temperature. Detailed records of arrival and expiry dates, and whether it needs to be aliquoted to maintain efficacy by avoiding ffeeze-thaw effects, should be kept. Attention to these details will preserve the efficacy of the antibody and its shelf hfe, prevent damaging freeze-thaw cycles, and will make optimization of the immunohistochemical procedures easier, ft is often necessary to aliquot antibodies, especially when storage below 4°C is required. 

Taschini, P. and MacDonald, D. (1987) Protease digestion step in immunohistochemical procedures ficin as a substitute for trypsin. Lab. Med. 18, 532-536. 

The presence of this artifact poses a distinct risk of its being interpreted as positive staining, as the artifact can be intense and may be precisely located in the cells of interest with a clean background. It is known that liver and kidney can retain high amounts of retrievable biotin-avidin activity in neoplasms. The need for adequate controls or biotinblocking procedures is obvious when histochemical or immunohistochemical procedures are used. Negative controls facilitate identification of such nonspecific staining. 

A minor disadvantage of the modified EnVision system is that it requires primary antibody concentrations four- to tenfold higher than those used in the conventional immunohistochemical procedures. Another limitation of this modified method is that only two slides with two sections each can be processed at any one time. 

Additionally, the principle of garbage-in, garbage-out cannot be overstated. There is no point in attempting to quantify IHC slides unless one is sure that the immunohistochemical procedure has been produced in a reproducible fashion. Automation and standardization in the process of preparing the IHC slides are very important components for successful image analysis. 

TABLE 10.2 Theoretical amplification and maximum test article concentration for various immunohistochemical procedures... 

Rickers RR, Malinisk RM. Intralaboratory quality assurance of immunohistochemical procedures Recommended practices for daily application. Arch Pathol Lab Med. 1989 113 673. 

OhsawaM, Naka N, Tomita Y, etal. Use of immunohistochemical procedures in diagnosing angiosarcomas Evaluation of 98 cases. Cancer. 1995 75 2867-2874. 

Discussed herein are the general requirements of the immunohistochemistry laboratory for compliance with CLIA-88. In addition, a troubleshooting guide for immunohistochemical procedures is included at the end. 

Proteins are usually detected by immunohistochemical procedures. Some proteins can be detected directly (e.g. by their errzymatic activity). However, the number of detected proteins depends on the availability of specific antibodies and often determined by the quality and the quantity of the surgical specimen. Immunohistochemical procedures discussed in the prior section have the advantage that the morphology of the tumor is still conserved and can be studied simultaneously with additional information regarding cellular antigens and their localization. In addition to immunohistochemistry, modern proteomics offers new approaches to detect proteins by western blotting, 2-dimensional gel electrophoresis or by protein arrays. 

Immunohistochemical Procedures for Light Microscopy The immunohistochemical staining using a biotin-streptavidin-peroxidase complex was performed according to the... 

Incubate the sections in the secondary antibody conjugated to the desired fluorochrome (see Note 47) and proceed with standard immunohistochemical procedures. Fig. 2B demonstrates the appearance of an immunohistochemically stained lacZ-positive cell. 

For storage of vibratome sections, as well as for the immunohistochemical procedures, we have found it very convenient to use 16-microwell plates... 

For monoclonal antibodies, the immunological properties of the antibody should be described in detail, including its antigenic specificity, complement binding, and any unintentional reactivity and/or cytotoxicity towards human tissues distinct from the intended target. Such cross-reactivity studies should be carried out by appropriate immunohistochemical procedures using a range of human tissues. 

For the calbindin immunohistochemistry described in the present example a standard immunohistochemical procedure is used. 

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