Immunofluorescence (IF/ICC) Troubleshooting: Why the immune fluorescence result has high background?

If your immune fluorescence results have high background, the main possible reasons are as follows.
1. Whether the antibody concentration use in test is too high. Select the optimal concentration and incubation time.
2. The incubation condition is not inappropriate. For example, the incubation time is too long or incubation temperature is too high.
3. Non-specific blocking is not sufficient. The blocking buffer is not right or contaminated. You can increase the blocking incubation time or changing blocking buffer.
4.The second antibody can binding to other things beside primary antibody which can lead to non-specific binding. The second antibody should be changed and prepared freshly.
5. The fixation procedures are too strong which can lead to the modification of antigen epitopes, then the antibody can binding to the target antigen.
6. The washing step is not done sufficiently. The immune fluorescence need enough washing in each step. Extensive washing with PBS is necessary.