Eclipse 90i

Ring glands were fixed in 4% formaldehyde for 20 minutes and washed three times in PBS for 5 minutes each. Samples were stained with 50 µg/ml of filipin (Sigma) for 1 h or incubated in an Oil Red O (Sigma) solution at 0.06% for 30 minutes. After incubation tissues were washed twice with PBS before mounting in Fluoromount-G. Samples were analyzed by conventional epifluorescence with a Nikon Eclipse 90i microscope or with a Nikon Eclipse 90i confocal microscope. Images were processed using Adobe Photoshop CS4 and Adobe Illustrator CS4.

In vivo, biodistribution of nanoparticles was investigated using FITC-labeled nanoparticles. The FITC-labeled nanoparticles were prepared by following the method described previously^{55 (link)}. Five mosquito larvae were placed in each well of a 24-well plate containing 1 ml of nuclease-free water. FITC-nanoparticles were evenly mixed with mosquito larval food and embedded in 1% melted agarose. This food pellet was fed to mosquito larvae under dark conditions. After 24 h, the whole larvae were viewed under 4X magnification of fluorescence microscope (Nikon ECLIPSE 90i). For biodistribution studies, mosquito larvae were dissected, and the alimentary canal and fat bodies attached to the epidermis were collected and washed with 1X PBS. The tissues were visualized under 10X magnification of a fluorescence microscope (Nikon ECLIPSE 90i).

To assess downstream targets of ROS, membrane disruption, and DNA damage, in C. difficile 2015, bacterial cells were washed thoroughly in PBS and adjusted to OD_600 nm of 1 in sterile anaerobic PBS and treated with 10 mM reuterin or 10 mM reuterin and 20 µM glutathione. As a positive control, C. difficile was also incubated with 500 μM H₂0₂. All cultures were incubated for 1 hr at 37ºC anaerobically. For membrane staining, C. difficile was incubated with 5 µg/ml FM 4–64FX (ThermoFisher #F34653) for 1 min on ice. Treated cells were then centrifuged at 5,000 x g for 5 min and fixed in 4% paraformaldehyde (PFA) at room temperature for 1 hr. Bacterial cells were counter-stained with Hoechst 33342 (Invitrogen) for 10 min at RT. Fixed bacteria were mounted on slides with Fluoromount Aqueous Mounting Medium (Sigma-Aldrich # F4680) and imaged on a Nikon Eclipse 90i. For DNA damage, C. difficile was incubated with 4% PFA at room temperature for 1 hr and dried on slides. Cells were permeabilized, treated with TUNEL staining reagents as previously described^{81 (link)}, and imaged on a Nikon Eclipse 90i. TUNEL stained images were quantified using FIJI (formerly known as ImageJ, National Institutes of Health) software by tabulating the mean pixel intensity in five regions per slide (n = 2 slides/experiment; performed two independent times).

Images were obtained using either a Nikon A1R spectral confocal mounted with either Plan-Apochromat 20x/0.8 WD = 0.55 M27 and Plan-APO 40x/1.4 Oil DIC (UV) VIS-IR objectives and ZEISS LSM 800 mounted with 20 × 0.5 CFI Plan Fluor WD:2.1 mm or 40 × 1.3 CFI Plan Fluor DIC WD:0.2 mm objectives for confocal acquisitions, from the University of Geneva bioimaging facility, while epifluorescence images were obtained on an Eclipse 90i Nikon epifluorescence microscope with a 10x, 0.44 micron/pixel objective.

pH7-WGY-AGO1 (pAGO1:YFP-AGO1) [26 (link)], 35S:HESO1-YFP [17 (link)]and 35S:HESO1-RFP [24 (link)] were previously described. pURT1:URT1^P618L-GFP was generated the same wasy as pURT1:URT1-GFP except that a hen1-2heso1-2urt1-3 genomic DNA was used as a template. Respective constructs were expressed in N. benthamiana leaves for 40~48hr and subjected to imaging using a Nikon A1 confocal mounted on an Eclipse 90i Nikon compound microscope.