Immunohistochemistry test antibody selection and common staining methods

I. Immunohistochemistry (IHC)
Immunohistochemistry is the use of the principle of specific binding of antigen-antibodies to determine the antigen (polypeptide and protein) in tissue cells by chemical reaction to develop the color of the labeled antibody (fluorescein, enzyme, metal ion, isotope). It conducts research on positioning, qualitative and quantitative. Immunohistochemical staining technology not only has high sensitivity and specificity, but also combines morphological changes with functional and metabolic changes to locate some proteins and peptides directly on tissue sections, cell smears or cultured cell slides. The presence of matter, and accurate to the level of subcellular structure, combined with electronic computer image analysis system or laser scanning co-aggregation microscopy and other techniques, quantitative analysis of the substance to be tested.
What are the tissue and cell specimens used in immunohistochemistry ?
The experiment used mainly two kinds of tissue specimens and cell specimens. The former included paraffin sections (pathological large pieces and tissue chips) and frozen sections, which included tissue prints, cell slides and cell smears. Paraffin section is the most common and basic method for making tissue specimens. It is good for tissue morphology and can be used for continuous sectioning, which is beneficial to various staining observations. It can also be archived for a long time for retrospective study. Paraffin section preparation process Antigen exposure in tissues has certain effects, but antigen retrieval is possible, which is the preferred method for making tissue samples in immunohistochemistry.
Why do paraffin sections need to be repaired? What are the methods ?
The paraffin section specimens were fixed with formaldehyde, so that the intracellular antigen formed an aldehyde bond or a carboxy-methyl bond to block part of the antigenic determinant, and at the same time, cross-linking between the proteins made the antigenic determinant concealed. Therefore, in the IHC staining, it is required to perform antigen repair or exposure first, that is, the cross-linking formed between the molecules at the time of fixation is destroyed, and the original spatial form of the antigen is restored.
Common antigen repair methods include microwave repair, high pressure heating, enzymatic digestion, and boiled heating. The commonly used repair solution is a 0.01 mol/L citrate buffer at pH 6.0.

Second, the selection of antibodies for immunohistochemistry experiments
1, primary resistance selection points
(1) Select monoclonal or polyclonal antibodies. A specific antibody produced by a clone is called a monoclonal antibody, and a monoclonal antibody can be specifically targeted to a single specific antigenic determinant, just as a missile accurately hits a target. On the other hand, even with the same antigenic determinant, antibodies can be produced from several clones in the body, forming several monoclonal antibody hybrids called polyclonal antibodies. In the antigen-antibody reaction, the general monoclonal antibody is specific, but the affinity is relatively small, and the sensitivity of the detection antigen is relatively low; while the specificity of the polyclonal antibody is weak, the affinity of the antibody is strong, and the sensitivity is high, but non-specific staining is prone to occur ( Can be avoided by blocking etc.).
(2) Source of species. Generally, rabbit-derived antibodies are mostly polyclonal; while mouse-derived antibodies are mostly monoclonal, but there are also others. This article is mainly to match the source of the secondary antibody in the back.
(3) The purpose of the experiment is to detect what kind of antigen, ie species reactivity. This is very important, as indicated in the general instructions, such as mouse Ms, rat Rat, human Hum and so on.
(4) Can you do immunohistochemistry? Generally, the first-resistance instructions will be marked as WB, IHC, ICC, IF, etc. It is recommended to select the indicated antibodies, as they are generally proved by the article.
(5) Test specimen type. For the detection of paraffin sections or frozen sections, antibodies that can generally be used for paraffin sectioning may be used to detect frozen sections, but antibodies that can be frozen sections may not be able to detect antigens in paraffin sections.
(6) Manufacturer. The quality of the original antibody from foreign famous antibody manufacturers is generally no problem, especially the IHCPlusAntibodies series antibodies produced by Lifespan in the United States, all of which have been verified by pathological biopsy, 100% quality assurance, domestic Beijing Ximeijie Technology Co., Ltd. is available for sale, the price is a bit Expensive; the primary anti-working fluid used by domestic distributors is cheap, but sometimes the stability and repeatability of the results are slightly worse, and the repeatability of different batches is poor.
(7) The contradiction between price and quality. I personally think that the quality of an antigen pack may be better, because many primary antibodies avoid repeated freezing and thawing, and the stability in the diluent is poor (if the time is long), but the price is more expensive.

2, secondary resistance selection points
(1) Source of species. The source of the secondary antibody is determined mainly according to the source of the primary antibody. For example, the primary antibody is the source of the mouse, and the secondary antibody can be purchased from the anti-mouse (the sheep, the rabbit, etc.).
(2) Selection of markers. There are markers such as HRP, Biotin, and fluorescein. Generally, the SP three-step secondary antibody selects the Biotin-labeled secondary antibody to bind to the latter SP; and immunofluorescence staining requires the purchase of different fluorescein-labeled secondary antibodies, such as rhodamine, FITC, Cy3 and the like. This is mainly related to the presence or absence of the third and third antibodies.
(3) Choose IgM or IgG. Generally, the primary antibody is IgM, then the secondary antibody selects IgM; otherwise, the primary antibody is IgG, and the secondary antibody selects IgG.
(4) Manufacturers. General SP three-step method Our laboratory selects the secondary antibody staining kit of Zhongshan Jinqiao, which contains the secondary antibody of the working fluid, so the experiment only needs to explore the concentration of the primary antibody, the effect is OK; and the immunofluorescence secondary antibody I Generally choose Jackson ImmnoResearch, the effect is not bad.

Third, what are the commonly used staining methods for immunohistochemistry? <br>According to the different markers, immunofluorescence, immunoenzymatic labeling, affinity histochemistry, the latter is a substance with a certain degree of tissue composition Affinity-based detection methods. This method is more sensitive and facilitates the localization of trace antigens (antibodies) at the cellular or subcellular level, with biotin-avidin staining being the most common.
Immunohistochemical staining methods There are many IHC staining methods. The nature of the label can be divided into fluorescence method (fluorescein labeling), enzymatic method (horseradish peroxidase, alkaline phosphatase), immunization of gold and silver and iron. Marking technique, etc.; according to the dyeing step can be divided into direct method (also known as one-step method) and indirect method (two-step, three-step or multi-step method); according to the combination method can be divided into antigen-antibody binding, such as PAP method and labeling Labeled dextran polymer (LDP) method, and affinity linkage, such as ABC method, labeled streptavidin-biotin (LSAB) method, etc., LSAB is the most commonly used method.
There are many factors affecting the quality of immunohistochemical staining. In the experiment, attention should be paid to the organization and fixation of the tissue, the selection of good quality commercial antibodies, the appropriate selection and use of blocking and antigen retrieval methods, strict technical operation and control. False-negative reactions may occur due to decomposition of the antigen to be tested in the tissue, or low antigen content, improper use of the fixative, poor antibody quality, and poor dilution; conversely, due to the crossover of the antibody with the non-test antigen False positive results can also occur with reactions, or non-specific adsorption of antibodies to tissues and endogenous peroxidase (endogenous peroxidase). These can cause mistakes in judgment.

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