As the PLA technique is very sensitive, special care is needed to keep the incubation times and conditions equal for different samples. In addition, we draw attention on the fact that the number of dots may vary from one experiment to another. Obviously, the duration of each step is crucial and should be strictly respected. We have also observed a slight decrease of efficiency of the kit over time. To prevent enzyme activity degradation, enzymes must be kept at −20 °C and added just before applying the reaction mixture to the sample. To overcome the kit limitation, we also recommend the systematic inclusion of a positive control by a single protein recognition (e.g. detection of total RUNX1 using 2 RUNX1 antibodies) for each experiment. This positive control can be used to normalize PLA data, allowing comparison between different series of experiments.