Immunofluorescence (IF) is a common experiment in immunology experiments. For the experimental masters, it may be the effort of sprinkling water, and a “dazzling and beautiful” film will come out, but for the novice operator, it may be in the overall process. It is not very friendly, and there will still be situations such as fluorescent bright spots in the entire cell sheet, or weak or no signal, too few stained cells, or only the blue light of DAPI in the observation field, and the target fluorescence has almost no or no signal. If so, it might be very frustrating. Today, let’s walk into the world of immunofluorescence and see how to sort out our experiments.
Immunofluorescence is a technique established on the basis of immunology, biochemistry and microscopy. It is based on the principle of antigen-antibody reaction. First, a known antigen or antibody is labeled with a fluorescent group, and then the fluorescent antibody (or antigen) is used as a probe to check the corresponding antigen (or antibody) in cells or tissues. Fluorescence microscopy can be used to visualize the cells or tissues where the fluorescence is located, to determine the nature and localization of antigens or antibodies, and to determine the amount using quantitative techniques such as flow cytometry.
The experimental application of immunofluorescence is extremely wide, and it can determine various biologically active substances such as endocrine hormones, proteins, peptides, nucleic acids, neurotransmitters, receptors, cytokines, cell surface antigens, tumor markers, and blood drug concentrations. According to the diagnosis category, it can be further divided into infectious diseases, endocrine, tumors, drug testing, immunology, blood type identification, etc.
3. Experimental steps
The main steps of immunofluorescence experiments include cell sheet preparation, fixation and permeabilization, blocking, antibody incubation, and fluorescence detection. The preparation of cell sheets (commonly known as cell crawling) is the first step in an immunofluorescence experiment. The quality of the cell sheet is crucial to the success of the experiment. The reason is very simple. If the cells fall off, everything will be out of the question.
4. Possible reasons for experimental errors
(1)Eg – Fluorescent bright spots appear throughout the cell sheet?
The reason may be that the secondary antibody concentration is too high, and the secondary antibody concentration can be appropriately adjusted according to the recommended dilution and titer ratio of the secondary antibody.
(2)Eg – weak or no signal, too few stained cells?
It may be that the target protein is not expressed in the cells, or the cells expressing the target protein are too few, and the cell permeability is poor
(3)Eg- There is only blue light of DAPI in the observation field, and the target fluorescence is almost no or very weak?
This phenomenon is likely because the proportion of antibody is too low, the concentration of antibody needs to be increased, and the concentration of secondary antibody can be increased; the excitation fluorescence intensity of confocal is not large enough.