Difference Between Direct Immunofluorescence and Indirect Immunofluorescence



The principle of immunofluorescence cytochemical method is to label the known antigen or antibody with fluorescein according to the law of antigen-antibody reaction to make fluorescent antigen or antibody, and then use it as a probe to detect corresponding substances in tissues or cells. The specific types of methods include direct method, indirect method, sandwich method and complement method. Under a fluorescence microscope, the source, nature and location of the detected substance can be determined according to the fluorescence emitted by the complex formed.

1. Direct method: This is the earliest method. The basic principle is to use a known antibody to label fluorescein to become a specific fluorescent antibody. When staining, the antibody is directly dropped on the slide for incubation, so that it can directly bind to the antigen on the slide and can be directly detected under a fluorescence microscope. Observe and make judgments. Evaluation of this method: simple, easy to implement, high specificity, fast and convenient, and commonly used in the detection of several immunoglobulins in renal biopsy tissue and the detection of pathogens. But its disadvantage is that only one corresponding substance can be detected, the sensitivity is poor, and the effect is sometimes unsatisfactory.

2. Indirect method: The basic principle of this method is to use a specific antibody to combine with the antigen in the slice, and then use an indirect fluorescent antibody to combine with the previous antigen-antibody complex to form an antigen-antibody fluorescent complex. Under a fluorescence microscope, the detected antigen is determined by the luminescence of the complex. Evaluation of this method: due to the increase of fluorescein antibodies bound to the antigen-antibody complex, the emitted fluorescence brightness is strong, so its sensitivity is strong. At present, this method is widely used, and it is only necessary to prepare one species of fluorescent antibody, which can be applied to the labeling display of multiple primary antibodies.

3. If you have a primary antibody (fluorescein-labeled) against the antigen to be tested, and your antigen to be tested is also abundantly expressed, it is not a bad idea to do the direct method. However, if you do not have the above two conditions, it is recommended that you do the indirect method.

4. If you are doing cytoskeleton, then direct immunofluorescence. If it is general protein expression, it depends on whether the reagents are directly labeled; if not, only indirect fluorescence can be considered. Compared with indirect fluorescence, direct fluorescence can avoid non-specific staining, and of course there are fewer steps. I have done direct staining of the cytoskeleton and it works great.