What is immunofluorescence?
It is a set of diagnostic techniques that use fluorescent substances, fluorochromes, to detect the presence of an antigen or antibody in cells or tissues.
This technique is mainly used for the diagnosis of autoimmune diseases.
What does it consist of?
Immunofluorescence works in 4 key steps:
- Fixation. The location, composition and structure of the biological material are preserved.
- Permeabilization. Pores are produced in the cell membranes allowing the antibodies to enter the cell.
- Blocking. The objective is to prevent nonspecific interactions of the antibodies with the biological material to be analyzed. In this way, nonspecific labeling is reduced.
- Immunodetection. In this last step two types of immunofluorescence are differentiated: direct or indirect.
Immunofluorescence: direct and indirect
Immunofluorescence is mainly used to diagnose autoimmune diseases. That is, those in which the body’s own defenses attack an organ of our organism.
In the case of direct immunofluorescence the fluorochrome is directly conjugated to the primary antibody.
In indirect immunofluorescence, on the other hand, unlabeled specific antibodies bind to the antigen and, in a second step, the labeled antibody is added as a fluorochrome.
Unlike direct immunofluorescence, the fluorochrome is conjugated to a secondary antibody directed against the primary antibody.
This test also has a higher sensitivity than direct immunofluorescence.
What is it used for?
It is used to determine the presence and distribution of proteins, carbohydrates or small molecules and can be used on tissue sections, single cells and secretions with cells in suspension. Immunofluorescence can be combined with other techniques that do not use antibodies.
Finally, it should be noted that it is a technique that can be applied to both biological and non-biological samples.