Immunofluorescence (IF/ICC) Troubleshooting: Why the immune fluorescence result is no staining?

If your immune fluorescence results are totally negative, the main possible reasons are as follows.
1. The primary antibody is compatible to the secondary antibody. For example, the primary antibody were raised in mouse, the second antibody is goat-anti-rabbit antibody. The results are certainly negative.
2. The target molecules are not present in the cells. To confirm whether the negative result is based on this factor, you can design a positive control cell with expression of target molecules.
3. The antibody has been damaged because improper storage or transport or other factors. If the antibody has other application, you can do the test to confirm whether the antibody was damaged. For example test the antibody by IHC, WB or FC.
4. The antibody bound to the target molecules is not enough. In next test the high concentration should be used and the incubation should be longer.
5. The target molecules are may not abundant in the cell. Thus, the amplification step should be tried to maximize the signal.
6. In the process of experiment, the second antibody has exposure to light. The second antibody has been labeled with fluoresce
7. When the buffer is contaminated with bacteria that damage the phosphate groups on the target molecules.