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Correct denaturation and hybridization temperatures are paramount for successful ISH. Regardless of the probe or target being RNA or DNA, it is vital that the probe is properly denatured (normally at 95°C) just prior to application onto the sample and chilled immediately on ice to prevent reannealing. Hybridization should be carried out on pre-warmed specimen at the desired hybridization temperature (commonly between 55-62°C) in a humidified, heated hybridization chamber. Displayed temperatures on devices might vary largely from actual temperatures, it may therefore be advisable to check actual temperatures with a sensitive thermometer. If a heating plate is used, it should be ensured that the plate is heated evenly and the plate surface is at desired temperature.