What are the advantages and disadvantages of varying incubation time and temperature for immunofluorescence staining of tissue?

Higher incubation temperature and shorter incubation time can reduce background fluorescence but can increase non-specific binding. Generally, it is not recommended for secondary antibodies to incubate as long as the primary antibodies due to the high background. Lower incubation temperature and longer incubation time can lead to higher background but less non-specific binding; it is also better for thicker tissue sections since longer incubation time allows better antibody penetration and therefore better signal.