The background intensity and specific-staining intensity are determined by conditions of DAB incubation. The developing time is not fixable and should be controlled under the microscope. After a clear signal of specific staining appears washing steps can be performed. If the DAB developing time was too short (10-60s), it might be due to a high antibody concentration or a long incubation time. Otherwise, a long developing time might be a result of a low antibody concentration or a too short incubation time.
If a short developing time with a high background, it might be due to an insufficient blocking. Similarly, if the developing time is too long, it might be due to a too long blocking time.