Szx10 stereomicroscope

Prior to treatment with CNTs, mature desiccated rice seeds were surface sterilized in a 70% ethanol solution for three minutes, followed by a 4.5% bleach wash for 35 min [38 (link),39 (link)]. Seeds were thoroughly washed five times with autoclaved deionized water to remove bleach. To initiate germination, surface-sterilized seeds were placed in a petri dish, covered with sterilized water, and incubated overnight at 29 °C.
Mature embryos were excised from sterilized seeds. Shoot tips were cut away to reveal SAMs, and the embryos were cut away from the endosperm using a fine-point scalpel. Such methods have been previously employed to facilitate the production of transgenic plants [40 (link),41 (link)]. Excised embryos were placed in a 0.6 M mannitol osmotic solution for two hours prior to drying on sterile filter paper and submergence in pDNA:CNT solutions at varying ratios.
Plasmids were covalently attached to positively charged CNTs at least 30 min prior to use. Embryos were vacuum-infiltrated at 500 mm Hg for five minutes in CNT solution before storing at 29 °C on a 14-h light, 10-h dark cycle. Images were taken at 24-h intervals by Olympus SZX10 stereomicroscope (Waltham, MA, USA).

Visualization of β-galactosidase activity with X-gal and endogenous GFP or YFP fluorescence in whole mounts has been described previously^{10 (link),33 (link)}. For X-gal staining, whole mount tissues were fixed for 30 min on ice with 4% PFA in PBS and washed with buffer A (2 mM MgCl₂ and 5 mM EDTA in PBS) twice for 5 min at room temperature. After incubation with buffer B (2 mM MgCl₂, 0.01% sodium deoxycholate and 0.02% Nonidet P40 in PBS) for 30 min, β-galactosidase activity was visualized with buffer C (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and 1 mg/ml X-gal in buffer B) at 37 °C overnight. Images of X-gal staining were acquired on an Olympus SZX10 stereomicroscope with a DP71 digital CCD camera. Fluorescence wholemount images were collected as z-stacks and projected into a single image using a Leica SPE confocal microscope.

Two-day-old common bean seedling were transferred into modified nitrogen-free Fahräeus medium plates (Catoira et al., 2000 (link)) and inoculated with 1 ml suspension (O.D.₆₀₀ = 0.3) of rhizobia WT (WT/pGUS), mutant (ΔRetPC57/pGUS), or the complement (ΔRetPC57/pGUS-pc57) strains. After four and six dpi, the zone of the root susceptible to rhizobial infection (hereafter referred as susceptible zone) were collected and immersed in GUS staining solution [0.05% 5-bromo-4-chloro-3-indoxyl-β-D-glucuronic acid (X-gluc), 100 mM sodium phosphate buffer (pH 7), 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 10 mM Na₂EDTA and 0.1% Triton X-100] and incubated for 3 h at 37°C. To evaluate the rhizobia adhesion to the common bean roots, pictures of susceptible zones showing blue color were captured using a bright field SZX10 stereomicroscope (Olympus) equipped with an Olympus UC50 camera (Olympus). To quantify the number of rhizobia-induced root hair deformation events, susceptible zones were examined under bright-field microscopy (Velaquin) equipped with an 18 Mega-Pixels Digital Camera with Aptina CMOS Sensor (Velaquin). For these experiments, four biological replicates, each one with six roots from different seedlings, were included.

Field surveys were carried out in the highlands of the Central Andes since 2005; collected material was deposited at B, CUZ, F, HSP, HOXA, HSP, HUSA, HUT, K, MO, MOL, RO, and USM, and it was useful to carry out necessary morphological comparison among the various taxa, just to verify the preliminary taxonomic value of them. In addition, herbarium specimens were also examined. Herbaria checked were: B, CONC, COL, CUZ, E, F, FI, G, GH, GOET, HSP, HOXA, HSP, HUSA, HUT, K, LIL, LP, LPZ, MO, MOL, MOQ, MPU, NY, P, PRC, S, SGO, SI, U, US, USM, and WAG (acronyms according to Thiers [21 ]). Relevant literature (protologues included) and online databases were analyzed [10 ,11 ,12 ,22 ,23 ,24 ]. The macro-morphological features described are based on detailed field notes and photographs made from fresh and dry material. The characters (both vegetative and sexual) of the new taxon and the related species Paronychia hieronymi Pax were studied under an Olympus SZX10 stereo microscope and an NSZ-405 1X-4.5X stereo microscope, followed by the morphological analyses of the roots, stems, leaves, flowers, and fruits, both from freshly collected and dried herbarium material. The variability of the characters was examined by box plots, performed using the software NCSS 2007.

Paraffin-embedded sections of spikelet samples were prepared according to Kim et al. (2014 (link)), with minor modifications. The materials were fixed in FAA and stored at 4 °C for 24 h. The fixed spikelets were dehydrated in a gradient ethanol series, and were then incubated in 100 % ethanol overnight. Dehydrated spikelets were embedded in Paraplast Plus (Sigma). The transverse sections of each spikelet were stained with 0.5 % toluidine blue, and viewed using an SZX10 stereomicroscope (Olympus, Tokyo, Japan). For scanning electron microscopy analysis, spikelet hull and endosperm samples were processed according to Wang et al. (2015b (link)) and Li et al. (2014b (link)). Young spikelets were fixed in 4 % (w/v) paraformaldehyde and 0.25 % glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2, at 4 °C overnight. Fixed spikelets were dehydrated in a graded ethanol series, and 100 % ethanol was replaced with 3-methylbutyl acetate (Toriba et al. 2010 (link)). Milled rice grains were transversely cut in the middle with a knife and were coated with gold under vacuum conditions. Samples were dried at their critical point, sputter coated with platinum, and observed with the XL-30-ESEM instrument at an accelerating voltage of 5 kV.

One-cell stage Tg (fli1:EGFP) embryos were microinjected with morpholino. At 2 dpf, Tg (fli1:EGFP) embryos were dechlorinated with pronase (Sigma-Aldrich Inc., St. Louis, MO, USA) and anaesthetized. Trypsinized Hep3B_Lifeact-RFP cells were collected, counted, washed, and resuspended in 1× PBS. We injected 4.6 nL of single-cell suspensions which contained 200 Hep3B_Lifeact-RFP cells into each embryo at the middle of yolk sac. After xenograft, embryos were washed with PTU/E3 and incubated in a RI-80 low temperature incubator. Temperature was gradually increased from 28 to 37 °C in 2 days, and maintained at 37 °C until the end of the experiment.
For observation the angiogenesis and proliferation of Hep3B_Lifeact-RFP cells, 0–3 dpi embryos were anaesthetized, placed on a 1% agarose plate for microscopy under a SZX10 stereo microscope equipped with DP71 digital camera and U-LH100HG fluorescence light source with U-RFL-T power supply (Olympus, Shinjuku, Tokyo, Japan). Red and green fluorescent images were obtained. Images of Hep3B_Lifeact-RFP cells from 0-3 day-post-injection (dpi) were quantified for fluorescent intensity as a readout of tumor cell proliferation activity. Length and number of ectopic vessels from subintestinal vein (SIV) were determined as indices of angiogenic activity. All the images were quantified using Image J software.