Lsm 5 exciter

Plasmids harboring SrTPS1-YFP, SrTPS2-YFP, SrTPS3-YFP, SrTPS4-YFP, and SrTPS5-YFP constructs were transformed into Agrobacterium tumefaciens GV3101 strain by electroporation and grown on Luria Bertani (LB) plates containing 20 mg/L of rifampicin and 25 mg/L of spectinomycin.
Cultures obtained from the above transformation were infiltrated into 4-week-old N. benthamiana leaves using a needleless 1 mL syringe. The agro-infiltrated plants were maintained in long-day conditions (16 h light/8 h dark, 25 °C).
For subcellular localization, infiltrated N. benthamiana leaves were mounted on slides three days post infiltration (dpi) and imaged using an LSM5 Exciter (Carl Zeiss, Oberkochen, Germany) confocal scanning laser microscope with a standard filter set. Images were processed using an LSM Image Browser (Carl Zeiss, Oberkochen, Germany).
For in vivo characterization of SrTPSs, VOCs were collected from N. benthamiana leaves 3 dpi, as described in the extraction of essential oils from Stevia tissues.

Adult hermaphrodites and adult males propagated on 1/10 MEA-chloramphenicol plates were picked up and transferred into a droplet of 0.1 M NaCl in the well of an eight-well glass slide^{25 (link)}. The worms were cut near the anterior region with a razor blade to release the gonad. The 0.1 M NaCl was completely exchanged for −20 °C methanol by pipetting, the nematode was then incubated for 5 min, and then stained with 2 µg/mL of DAPI in phosphate-buffered saline (PBS) for 10 min. After washing twice with PBS, the worms were mounted with Vectashield onto glass slides (Vector Laboratories Inc., USA). DAPI-stained images were obtained with a confocal laser-scanning microscope (LSM5 exciter, Carl Zeiss).

Liver tissues were cryo-sectioned and processed for the immunofluorescence (IF) assay as described previously (Alam et al., 2012 (link)). RBC-EM ^DiD or PBS-injected mouse liver sections were stained with anti-rabbit CD68 (Abcam), followed by staining with goat anti-rabbit FITC (Abcam). Tissue sections were mounted using the VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA, United States). IF-stained sections were imaged under a confocal microscope (LSM 5 Exciter, Zeiss, Oberkochen, Germany). Total of six fields were counted by observers. Number of CD68 positive (CD68 +; green) cells were counted from RBC-EM ^DiD or PBS-injected mouse liver sections. Then the number of DiD positive (DiD +; red) with CD68 + or CD68 - cells were counted.

HK-2 cells were grown to confluence in transwell chambers (0.4-μm pore size, Transwell Permeable Supports, Cat No. 3460, Corning) for 3 days. On day 4, HK-2 cells were treated with vehicle or 10, 25, 50 or 100 nM siRNA for 72 h and fixed with 4% paraformaldehyde in PBS, pH 7.4 for 20 min at room temperature. After fixation, cells were washed twice in PBS and permeabilized with 0.3% Triton X-100 in PBS at room temperature for 15 min. Cells were washed and labeled with anti-claudin-2, anti-occludin or anti-ZO-1. After incubation, cells were washed in PBS and incubated with goat anti-rabbit IgG Alexa Fluor 488 secondary antibody (A11008, Molecular Probes) or donkey anti-mouse IgG Alexa Fluor 488 secondary antibody (A21202, Molecular Probes) for 2 h at room temperature. Immunolocalization used a laser scanning confocal microscope (Zeiss LSM 5 EXCITER, Jena, Germany) [11 (link)].

Full-length ORFs of CsTPS3GT, CsTPS4WS, and CsTPS5TH were cloned into pENTR vectors using the pENTR™/D-TOPO^® Cloning Kit (Thermo Fisher Scientific, Singapore) and transformed into XL1-blue competent cells. Plasmids from the positive clones were isolated and cloned into the destination vector pBA-DC-YFP [71 (link)], which contains the YFP in frame at the C-terminal and cauliflower mosaic virus (CaMV) 35S promoter, to generate CsTPS3GT-YFP, CsTPS4WS-YFP, and CsTPS5TT-YFP, respectively. The constructs were transformed into the Agrobacterium tumefaciens EHA105 strain by a heat shock method [72 (link)]. The transformed EHA105 cells were cultured at 28 °C and resuspended in a solution containing 100 µM acetosyringone, 10 mM MES (pH 5.6), and 10 mM MgCl₂. The above mixture was incubated at room temperature for 3 h and later infiltrated into N. benthamiana leaves using a 1 mL syringe. After 2 d, the infiltrated leaves were excised, and the fluorescence signals were observed under a confocal scanning laser microscope (LSM 5 Exciter, ZEISS, Jena, Germany). All constructs were verified by DNA sequencing.