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Principles and Experimental Procedures of Immunofluorescence

Principle of immunofluorescence

Immunofluorescence is a technique established based on immunology, biochemistry, and microscopy technology. It is a process in which an antigen or antibody, known as a fluorescent probe, is labeled with a fluorescence group, and then the corresponding antigen or antibody in cells or tissues is examined using the fluorescent antibody (or antigen) according to the principle of antigen-antibody reaction. Fluorescence microscopy is used to visualize the cells or tissues with fluorescence, to determine the nature and location of the antigen or antibody, and to determine the content using quantitative techniques (such as flow cytometry).

Steps of immunofluorescence test

The main steps of immunofluorescence test include preparation, fixation, permeabilization (or called tanslocation), blocking, antibody incubation, and fluorescence detection. Preparation of cell slides is the first step in the experiment, and the quality of the cell slides is crucial to the success of the experiment because if cell falling occurs, everything will be impossible.

The key to this step is the processing of glass slides and the vitality of cells. Some people have summarized many useful details or tips based on successful experiences, which are very worthy of reference. The most important thing for fixation and permeabilization is to select an appropriate fixing method and fixing agent according to the properties of the studied antigen. The choice of suitable fixing agent and process is critical to obtaining good experimental results.

The blocking and antibody incubation in immunofluorescence are similar to the same steps in other methods such as ELISA or Western Blot. The most important difference is that fluorescent antibodies are used in immunofluorescence test, so it is necessary to remember to avoid light. In addition, the selection of antibody concentration may be more critical. Finally, it is important to note that the cell slides labeled with fluorescence should be observed as soon as possible or preserved in a light-avoiding manner after fixation with mounting medium at 4℃ or -20℃, to avoid influencing the experimental results due to dissociation of the labeled protein or weakening of fluorescence.

Because there are many steps and it is not possible to distinguish non-specific recognition according to the size of molecular weight like in Western Blot while analyzing results, in order to obtain a perfect immunofluorescence testal result, in addition to requiring high-quality antibodies and repeatedly optimizing experimental conditions, rigorous experimental controls must also be established.

In summary, every step from sample processing, fixation, blocking, antibody incubation to final mounting and observation of immunofluorescence test is critical, and the quality of each step in the experimental process needs to be strictly controlled in order to ultimately achieve the experimental purpose.

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