Zoe flourescent cell imager

Salivary glands and midguts harvested unfed O. turicata nymphs, were fixed and permeabilized using the Fix and Perm Kit (ThermoFisher) per the manufacturer's instructions. Tick tissues were incubated in PBS in the presence of BSA (10 mg ml⁻¹) and rabbit serum as a negative control or with a 1:100 dilution of a universal NOS antibody (ThermoFisher) for 3 h. Tissues were then subjected to 3 × 0.5 ml washes in PBS and then incubated with a 1:200 dilution of the Goat anti‐Rabbit IgG secondary antibody Alexa Fluor 488 conjugate (ThermoFisher) and a 1:400 dilution of DAPI (ThermoFisher) for an additional 3 h at RT. Tissues were washed as described above and then visualised by fluorescent microscopy using the Blue channel (Ex_λ = 355 nm, Em_λ = 433 nm) and the Green channel (Ex_λ = 480 nm, Em_λ = 517 nm) of the ZOE Flourescent Cell Imager (Bio‐Rad).

ROS and RNS generated in O. turicata salivary glands and midguts were detected with the fluorescent probes 2,7‐dichlorodihydrofluorescein diacetate (DCF‐DA) and 4,5‐diaminofluorescein diacetate (DAF‐2), purchased from Cayman Chemical (Ann Arbor, MI). DCFH is readily oxidised by ONOO⁻ to the highly fluorescent product dichlorofluorescein (DHF). DAF‐2 reacts with NO in the presence of oxygen to produce triazolofluorescein (DAF‐2 T). Briefly, salivary glands and midguts dissected from stage 2 O. turicata nymphs were rinsed extensively in PBS, and transferred to individual 1.5 ml microcentrifuge tubes with 25 μM DCF‐DA or DAF‐2 for 10 min at RT. Following incubation, tick tissues were placed in individual pools of PBS on glass microscope slides, and imaged using the brightfield and Green channel (Ex_λ = 480 nm, Em_λ = 517 nm) of the ZOE Flourescent Cell Imager (Bio‐Rad).