Counterstaining visualizes and localizes the cell profile by hematoxylin. The time for counterstaining depends on the room temperature, freshness of staining solution, and localization of antigens (seconds to minutes). If target antigens are cytoplasmic proteins, the time of counterstaining can be relatively long. Otherwise, for nuclear proteins, the time should be relatively short.
If there was an excessive counterstaining, after washing with water slides can be treated with hydrochloric acid and alcohol for a considerable time (longer than seconds), then washing and following with ammonia treatment.