What are standard secondary incubation conditions? Can incubation be extended?

One-step secondaries: 20 min at room temperature in a humid chamber. Two-step secondaries: 10 + 10 min at room temperature or 37°C in a humid chamber. Do not extend incubation arbitrarily—over-incubation increases non-specific binding, background, or false positives. If antigen expression is low, you may extend by 10–15 min.