It is well known that immunohistochemical technology plays an important role in studying the development of tumors and performing benign and malignant classification and differential diagnosis. Therefore, it is necessary to be cautious in doing such experiments, especially immunohistochemical staining experiments. The results are easy. Affected by certain factors. So, what factors are susceptible to immunohistochemical staining?
Experts pointed out that the results of immunohistochemical staining are closely related to three important factors: tissue fixation, antigen repair, antibody preservation and use.
1. Fixed
The commonly used tissue fixative is formaldehyde, and the specimen must be fixed in time, which is conducive to the preservation of the antigen, preventing the antigen from diffusing, losing or losing immune activity in the tissue cells, but the fixed time is preferably! "Hours, generally do not exceed" # hours, the longer the fixation time, the sensitivity of some tissue immunohistochemical markers will be significantly reduced. The reason is that aldehyde bond or carboxymethyl group is formed during formaldehyde fixation, and some antigenic determinants are blocked; cross-linking between protein and protein may also occur, and antigenic determinants may be blocked, so that many antigens are commonly used. $%, &$' (the immune response is significantly weakened or even disappeared, so that the enzyme label can not produce the correct results, so in order to obtain a good staining effect when dyeing, some antigens must be pre-repaired to further expose the antigen.
2, repair antigen
The externally examined tissues are subjected to formaldehyde fixation, dehydration, and waxing, and the antigen components in the tissues have been destroyed or blocked. In order to restore the antigenicity of the tissues and increase the sensitivity of the tissues to antibodies, it is generally necessary to repair the antigen. Some people use protease digestion, or microwave treatment, or pressure cooker treatment, so that the blocked antigen components are exposed to develop color. We used high-temperature and high-pressure treatment to slice, sliced ​​under weak acid and high temperature and high pressure, so that the blocked antigen was displayed, which improved the positive detection rate and positive intensity, and reduced the background coloration, so that the positive result was clearly distinguishable. Of course, different tissues, different antigens, the high pressure time used and the selection of the appropriate antigen retrieval buffer and its optimum. 'Equivalents have a great impact on the results.
3, the correct preservation and use of antibodies
Antibodies are the most basic reagents and materials for immunohistochemistry. They can be divided into primary antibodies and secondary antibodies. Because antibodies are composed of proteins, improper storage or use, not only will waste, but the reagents will have false negative results. The antibody and the 6 and 7 kits should be stored in a low-temperature refrigerator. Use one to take one. For one-time use of the antibody, it can be stored in the #8 refrigerator instead of the ice compartment, because the temperature there is + Below 8, the antibody will freeze quickly and will dissolve again when used again. In such a few freeze-thaw cycles, antibody titers will drop dramatically and fail. For some near-term failure periods or expired periods, and some appropriate increase in working concentration, correct results can also be made to avoid loss and waste.
Of course, the results of immunohistochemical staining affect , in addition to the above, there are other factors, such as slides need to use anti-offset, container cleaning treatment to reduce pollution. The time of antigen and antibody action needs to be adjusted. We extend the time of the primary antibody and find that the effect is better. Winter placement) 9 8 incubator incubation, shortening the duration of action, as well as the length of color development time, slice thickness is related.
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