According to the steps of experiment, immunofluorescence includes preparation of cells or tissues, fixed cells or tissues, permeabilized, closed and incubated with anti incubation second antibody, mounting and imaging steps.
Comparison between direct and indirect methods:Direct method is less sensitive, but the sensitivity is not high, and the indirect method, because of the surface of the antibody immunoglobulin molecules, can be combined with a number of fluorescent antibody molecules, and play a role in amplification, the sensitivity is higher than the direct method 5~10 times. There are many factors that are involved in the process of non - specific fluorescence, which is more than the direct method.
Every step of the immune fluorescence is critical, and every step error is either false or unreliable. So in general, laboratories or companies will be strictly regulate the operation of the immune fluorescence steps and processes, every step to ensure that no error, to get a good result .