The wash buffers should be brought to room temperature before use, as low temperature slows down the enzymatic reactions. The slides were washed four times, 5 min each in a staining jar under gentle shaking (60–90 rpm/min) at room temperature: first with 1X PBS/0.5% tween-20, second with 1X PBS/0.2% saponin/0.5% tween-20, third with 1X PBS/0.5% tween-20, and fourth with 1X PBS. The 5X Ligation buffer was diluted in water for a final concentration of 1X and the ligase (1 U/μL) diluted in 1:40 in the ligation 1X solution. Fresh dilutions should be prepared just before use. Samples were incubated with 15 μL of ligase solution for 1 h at 37 °C in the humidity chamber.