Immunocytochemistry is often applied to the identification of constructed and short-term cultured cells, the detection of cell-specific antigenic components, the study of cellular characteristics, and the screening and identification of monoclonal antibodies. Immunocytochemistry is a new method that has been developed in recent years and is still under constant improvement. It has the advantages of accurate and reliable characterization, no damage to the intrinsic structure and composition of cells, and can be combined with other morphological observation methods for research. Source of content: Tissue Culture and Molecular Cytology Techniques. (Beijing Publishing House)
Operation method
Immunocytochemical techniques for observing cellular components
Principle
Immunocytochemistry is a technique developed on the basis of cytochemistry and absorbing the theories and techniques of immunology. Its basic principle is to use fluorescein or enzymes and other markers or chromophores to label specific antibodies, and then through the antigen-antibody reaction in immunology to combine with the corresponding antigens in the cell to form the antigen-antibody complexes with fluorescein or chromophores, and therefore the presence of the complexes can be observed by fluorescence microscope or common optical microscope to achieve the purpose of detecting the antigenic substances in the cell. Therefore, the presence of the complex can be observed by fluorescence microscope or ordinary light microscope, and the purpose of detecting the antigenic substances in the cell can be achieved. Cellular immunochemistry is a comprehensive technology, including specimen preparation (preparation, storage, fixation, etc.), staining methods and results of analysis and judgment, non-specific staining mitigation, elimination and so on.
Materials and Instruments
Cells Move I. Specimen preparation Caveat 1. Live cell specimens should be fixed immediately after preparation to prevent cell autolysis.Maintain its inherent organizational structure, preserve the antigenicity of the cells, so that the antigen is not inactivated, does not dispersion and to ensure that the immunohistochemical localization is accurate.The immunohistochemical localization is accurate. After fixation, the specimen must be washed thoroughly and the excess fixative removed, otherwise it will affect the immunohistochemical staining effect.The color effect will be affected. 2. The general fixative is suitable for room temperature (22 ℃), and the fixation of certain enzymes should be carried out at 37 ℃. Fixation timeThe fixation time varies depending on the fixative, temperature and antigen composition. If the time is too short, the fixation will be insufficient, affecting the antigen preservation.However, the longer the time, the better, the longer the time will also have an impact on the antigen, cell morphology, thus affecting the immunohistochemistry effect, so it should beImmunohistochemistry effect, so it should be mastered appropriately. Fixation time: alcohol, acetone 5-15 minutes, paraformaldehyde 3-5 minutes, formalin 10-15 minutes.minutes, formalin 10-15 minutes. For more product details, please visit Aladdin Scientific website.
Formalin Acetone Paraformaldehyde
Coverslips Culture flasks Culture plates
Cells can be cultured directly on coverslips, in culture flasks or on culture plates, or a certain amount of cells can be collected and centrifuged A certain amount of cells can also be collected and centrifuged to make a smear.
Fixation and fixation system
For live cell specimens, the following fixatives are commonly used.
1. formalin
10 % formalin or 10 % neutral formalin, of which 10 % neutral buffered formalin has the best fixation effect and is most commonly used. The best effect of fixation of 10 % neutral buffered formalin, the most commonly used.
2. acetone class
Pure acetone fixation effect is good, the impact on the antigen is also smaller, 95 % ethanol fixation effect is similar to acetone but not as good as acetone. Such as adding 1 % glacial acetic acid in ethanol can enhance the fixation effect, adding a small amount of chloroform can be conducive to the penetration of antibodies.
3. 4 % paraformaldehyde Dissolve 40 g of paraformaldehyde in 0.1 M PBS 1000 ml, heat to about 60 ℃, stirring while warming until transparent. (generally need to add a little 1 M NaOH to make the solution clear). This fixative is mild and suitable for the long-term preservation of specimens. This fixative is milder and suitable for longer term storage of specimens.
At present, there is no universal fixative that can satisfactorily fix various antigens. In comparison, neutral buffered formaldehyde is In comparison, neutral buffered formaldehyde is a more versatile fixative. If necessary, a variety of fixatives can be compared, from which the best fixative for a particular antigen can be selected. If necessary, a variety of fixatives can be compared, from which the fixative with the best effect on a certain antigen can be selected.