The next day, the slides were washed three 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 (#P9416, Sigma Aldrich), second with 1X PBS/0.2% saponin/0.5% tween-20 and the third with 1X PBS/0.5% tween-20. Then, secondary antibodies conjugated with oligonucleotides (PLA probe anti-species 1 MINUS and PLA probe anti-species 2 PLUS) were diluted 1:5 in preblocking buffer and 15 μL/spot was applied for 1 h at 37 °C in a humidity chamber.