Mzfiii

Postnatal day 18 (P18) and P21 old mice were terminally sedated using a 1 : 2:1 solution of fentanyl citrate and fluanisone anaesthesia (Hypnorm, VetaPharma), ddH₂O and midazolam hydrochloride (Hypnovel, Roche) at a dosage of 5 µl per 1 g. After sedation occurred, the tympanic membrane was carefully punctured and mice were injected with a 1% aqueous solution of the fluorescent tracer Fluorescein (Sigma) into both middle ear cavities and left in a prone position. Mice were culled by cervical dislocation after a validated time of 5 min. To visualize the soft palate and nasopharynx to pharynx orifice, the mandible and tongue were dissected and photographed in a supine position to allow a ventral view of the palate using a dissection microscope (Leica MZFiii) and Leica DFC300 camera with fluorescence.

Fish were briefly anaesthetised in tricaine solution (0.1 g l⁻¹ MS-222), placed onto a Petri dish while immersed in the tricaine solution and imaged using a Nikon COOLPIX5400 camera attached to a brightfield microscope (Nikon SMZ1500). Each fish was imaged using three consecutive photo shots that were later merged together using Adobe Photoshop automerge function. For fluorescent imaging, anaesthetised fish were imaged under a Leica MZFIII stereomicroscope equipped with a Retiga R1 camera operated via µManager software (Edelstein et al., 2010 (link)). Images were taken using GFP and mCherry fluorescent filters. The fluorescent images were processed using Fiji software version 2.1.0 (Schindelin et al., 2012 (link)) for pseudocolouring and adjusting minimal and maximal intensity.
For zebrabow live imaging, anaesthetised fish were imaged under a Leica M205FCA stereoscope equipped with Leica K8 camera and operated via LASX software (Leica). Images were taken with a 0.63× objective at 1× or 2.5× zoom using CFP, YFP and mCherry fluorescent filters and processed as above. As shown in Fig. S1B, the CFP channel showed a certain level of autofluorescence, most prominent in the creases of jaw and gills, that was not considered as positive signal.

GFP fluorescence of the whole plants or the intact leaves and flowers was viewed with a long-wavelength UV light and was photographed directly using a Canon EOS 600D digital camera. Images of fluorescence distribution in hand-cut cross and longitudinal sections of petioles and roots from LIYV-L2C_GFP infected N. benthamiana plants were taken with a Leica MZFIII fluorescence stereomicroscope (Leica Microsystems, Wetzlar, Germany) provided with UV illumination and a GFP filter. To observe the magnified leaf veinlets, the epidermal cells were removed from the lower side of leaf tissue and mounted in water. GFP fluorescence was observed using a Leica DM5000 B fluorescence microscope (Leica Microsystems).