Lsm 510 confocal scanning microscope

Taste cells dissociated from taste buds in fungiform papillae (see ''Single Taste Cell Isolation'') were placed on coverslips, allowed to settle for $30 min, and then fixed with 4% PFA-PBS for 30 min at room temperature. Cells were washed in PBS for 3 3 5 min, and subsequently incubated overnight at 4 C with sheep anti-NTPDase2 antibody (1:10,000, AF5797, R&D systems, Minneapolis, MN, USA) in PBS containing 3% normal donkey serum. The next day, cells were washed in PBS for 3 3 5 min, incubated at room temperature for 1 h with biotin-conjugated donkey anti-sheep IgG antibody, washed in PBS for 3 3 5 min, fixed again with 4% PFA-PBS for 30 min at room temperature, washed in PBS for 3 3 5 min, and incubated at room temperature for 1 h with chick anti-GFP (1:1,000) and rabbit anti-RFP (1:1,000) antibodies in PBS containing 3% normal donkey serum and 0.1% Triton X-100. Finally, cells were labeled with Alexa Fluor-488 conjugated donkey anti-chick IgY and Alexa Fluor-546 conjugated donkey anti-rabbit IgG antibodies, and Alexa Fluor 647-conjugated streptavidin, and mounted in fluoromout-G (SouthernBiotech). Images of stained cells were captured with an LSM510 confocal scanning microscope (Carl Zeiss) using an EC Plan-Neofluar 40 3 /1.30 NA Oil objective.

The full-length cDNAs of LlWOX9 and LlWOX11 under the control of the 35S cauliflower mosaic virus promoter were cloned into the pCAMBIA 2300 vector using the pEASY ® -Basic Seamless Cloning and Assembly Kit (Transgen Biotech, China) . The sequences of the primer pairs used for amplification are shown in Table S3. The resulting plasmids were transferred into Agrobacterium tumefaciens strain GV3101. Agrobacterium cells were collected and suspended in infiltration buffer (10 mM methylester sulfonate, 10 mM MgCl 2 , and 150 mM acetosyringone, pH 5.7) at OD 600 =0.8 and infiltrated into Nicotiana benthamiana leaves. 3 days after infiltration, the leaves were harvested and treated with 0.5 mg/ml DAPI (4ʹ,6-diamidino-2-phenylindole; Sigma). A Zeiss LSM 510 confocal scanning microscope was used to collect images.

Leaf axils in S1-S5 were fixed with FAA, and after dehydration, clearing and embedding, paraffin sections of the leaf axils were sliced at a thickness of 0.8 μm. The obtained slides were rehydrated with xylene, digested with protease K (20 μg/mL) at 37°C, blocked with a 3% methanol-H2O2 solution for 25 min, with avidin (0.07%) at 37°C for 25 min and with biotin (0.005%) at 37°C for 15 min. Hybridization with the probes was performed at 37°C overnight in a moist chamber. After hybridization, the slides were washed in 2× SSC for 10 min, three times in 1× SSC for 5 min and once in 0.5× SSC for 10 min at 37°C. The avidin-labelled probe was detected with a streptavidin Alexa Fluor 405 conjugate (1:250) (Invitrogen). Antibodies were diluted in PBS containing 3% (w/v) BSA, and the slides were incubated with the antibodies for 30 min at 37°C. After antibody incubation, the slides were washed three times with 4× SSC containing 0.1% Tween 20, stained with 100 ng/mL DAPI in PBS for 30 min and dehydrated with ethanol. Confocal images were obtained using a Zeiss LSM 510 confocal scanning microscope.