Ec-LacZ dsRNA was labelled with the dye Cy3® [10 (link)] using the Silencer™ siRNA labelling kit (Thermo Fischer Scientific, Waltham, MA, USA). Twenty-five each of pre-treated or untreated eggs were incubated in 2.5 µg/µL of Cy3® labelled Ec-LacZ dsRNA in 0.9% nuclease free NaCl at 4 °C for 24 or 48 h or at room temperature (22 °C) for 24 h. Incubated eggs were washed by dipping them 3 times into PBS-0.05% Tween (PBST) and once into PBS to remove excess dsRNA before examination under a Zeiss 780-NLO confocal microscope with a 20× Plan-Apochromat (NA 0.8) lens. Z stack images (4–8 slices) were captured. Cy3® was excited using a 561 nm laser attenuated 97% and emitted light (565–631 nm) was captured using a GaAsP array detector. Images are presented as a single representative slice through each egg processed using Zen software (Carl Zeiss AG, Oberkochen, Germany). Eggs with developed larvae were excluded, to eliminate false positive signals due to autofluorescence of the S. scabiei cuticle. The dsRNA uptake efficiency was determined as the percentage of fluorescent positive eggs of the total number of eggs incubated.
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