Then, the slides were washed three times, 5 min each in a staining jar under gentle shaking (60–90 rpm/min) at room temperature: 1X PBS/0.2% saponin/0.5% tween-20, second with 1X PBS/0.5% tween-20 and the third with 1X PBS. The amplification 5X was diluted extemporaneously in water for a final concentration of 1X and the polymerase (10 U/μL) diluted in 1:80 in the amplification 1X solution. Samples were incubated with 15 μL of amplification solution for 1 h 15 at 37 °C in the humidity chamber. The samples should be protected from light in order to avoid bleaching of the fluorophores.