Immunofluorescence / IF / ICC is a common laboratory technique widely used in almost all field of biology. There are two main methods with different principle for Immunofluorescence / IF / ICC that are indirect immunofluorescence and direct immunofluorescence. In indirect immunofluorescence, the labeled antibody is second antibody and the primary antibody specific for the target molecule is unlabeled. In direct immunoflurescence, the primary antibody specific for the target molecule is labeled. Indirect immunofluorecence and direct immunoflurescence have advantages and disadvantages. Indirect immunofluorecence method is used more frequently used than direct immunofluorescence.
The advantages of indirect immunofluorescence are high sensitivity, easy to change signal color based on changing second antibody which can be get commercially. The labeled second antibodies are conveniently obtained.
The disadvantages of indirect immunofluorescence are the potential cross reactivity, finding labeled primary antibody which is more difficult to get especially for multiple labeling experiments. On the other hand, the high background is hard to avoid when the target molecule has with endogenous immunoglobulin be tested.
There are some advantages for direct immunofluorescence. The process time of direct immunofluorescence is shorter than indirect immunofluorescence. It is necessary to do direct immunofluorescence when multiple antibodies from the same species. There are some disadvantages for direct immunofluorescence. The first is lower signal than indirect immunofluorescence. The second is that direct immunofluorescence has higher price than indirect immunofluorescence because of difficulties and less flexibility with the labeling procedure when commercially labeled direct conjugates are unavailable.