1. Washing is not sufficient uring the incubation step.
2. The sample is dried after antibody incubation. So it is necessary to keep moist samples before mounting.
3.Incubation of the antibody before the closure is not complete.For different experimental samples, you can try different sealing reagent.
4.You should choose appropriate fixed reagents and fixed method.
5.The samples are too thick or the sample has spontaneous fluorescence.