Immunofluorescence is one of the earliest developments in labeling immunoassay. It is a technology based on immunology, biochemistry and microscopy. It combines antibodies with some tracer substances and uses antigen-antibody reactions to organize or Localization of intracellular antigenic material. The immunofluorescence step includes cell fixation and permeabilization, blocking, incubation of primary antibodies, secondary antibodies, and the like. In addition to these basic steps, the following suggestions can help you get better experimental results. |
1, cell fixation and permeability <br> In order to achieve the best detection results, the cells need to be fixed and transparent. These steps are critical, and the cells and antigens need to ensure optimal structure and facilitate antibody binding to the antigen. Typically, this is achieved by the following steps: Cells are fixed with 2%-4% paraformaldehyde followed by permeabilization with 0.1% saponin or 0.02% Triton X-100. The former is a gentle treatment, but it may not be effective for nuclear antigens, and Triton is needed. When saponin is used for permeabilization, it should be noted that it causes reversible permeability of the cell membrane, that is, in addition to the initial phase of permeabilization, it is required to be permeabilized in each antibody incubation step. In addition, cells can be fixed and permeable with ice methanol to avoid the use of detergents. | 2, antibody specificity
Immunofluorescence requires the use of very specific antibodies that avoid high background and undesirable protein localization results. In most cases, the purification of antibodies works well, but the right controls can help pinpoint the antigen. The use of a secondary stain-only stain as a negative control is beneficial in reducing background interference. | 3. Appropriate antibody dilution ratio <br> Optimize staining by optimizing the antibody dilution ratio. Usually, 1 ug/ml of purified antibody or 1:100-1:1000 antiserum is sufficient to achieve specific staining results. However, under the premise of ensuring low background dyeing, the signal intensity can be increased by increasing the concentration. If the antibody is used for the first time or an antigen is determined, a concentration gradient experiment is strongly recommended. | 4. Optimize Buffers and Blockers <br>Although many antigens are well stained in common Buffers such as PBS, for some antigens of interest, replace buffers containing different ions, such as calcium, magnesium, and potassium. Etc., can bring a lot of improvement. Rockland offers optimized IHC blocking buffers, which are also suitable for fluorescent staining experiments. | 5, choose the right secondary antibody
If you need to do an immunization experiment, we strongly recommend that you choose a pre-adsorbed secondary antibody for a single staining experiment; if it is a double or even multiple staining, you must use a pre-adsorbed secondary antibody. At the same time, please choose the secondary antibody from the same species. | 6, using the appropriate cell density <br> select the appropriate number of cells for staining, when the number of cells is too large, the cell structure is not good, resulting in deep staining background, low cell density, will cause poor cell adherence, the state is not good. | 7. Multiple staining <br> When two different antigens are detected in the same sample, simultaneous staining can be performed with the respective antibodies, but the species of the two primary antibodies are required to be different, the markers are different, and the species of the secondary antibodies are different. Sources need to be consistent. | 8, reduce the background <br> high background is a common problem of immunofluorescence, to solve this problem, you can use the normal serum from the source of secondary antibodies instead of BSA as a blocking solution, reduce the antibody concentration, increase the number of washings, wash at least three times, five minutes each time, The recommended washing solution is PBS + 0.05% Tween. | 9, cover
As the final step in immunofluorescence, the refractive index can be increased to protect the sample. | 10, data analysis <br> When observing the whole sample, select representative cells for data acquisition and analysis. See: http://?utm_source=tips&utm_medium=email&utm_campaign=marker |
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