Powershot a620

GUS staining was carried out according to the method reported in Hu et al. [50 (link)]. GUS activity in primary root apices in 5-day-old DR5::GUS seedlings treated with 0, 25, 50, 100 μM Cd for 3–4 days on 1/2 MS plates. GUS-stained images were observed under a stereo microscope (ZEISS Stemi 2000-C) and photographed by a CCD camera (Canon PowerShot A620).
Confocal microscopy was performed using a confocal laser scanning microscope (PerkinElmer, Waltham, MA, USA, UltraVIEW^® VoX) according to the manufacturer’s instructions, excitation and emission wavelengths were 488 to 520 nm for DII-VENUS. Auxin signaling level in primary root apices in five-day-old Arabidopsis thaliana DII-VENUS seedlings treated with 0, 25, 50, 100 μM Cd for 3–4 days on 1/2 MS plates.

To characterize female development, mating experiments were carried out in triplicate between isolates of opposite mating types lacking the same step in the sphingolipid pathway. Conidia from the isolates were inoculated on two sterile 6 cm in diameter discs of Whatman 3 MM chromatography paper placed in a Petri dish with 5 mL of sterile synthetic crossing medium [41 ]. Whatman 3 MM paper serves as a carbon and energy source for the fungus. The strains were allowed to grow in the dark at room temperature for eighteen days. During this time, the fungus produced perithecia, the N. crassa female mating structure. Sterile water was added as needed to maintain a moist state in the filters. By eighteen days post-inoculation, wildtype perithecia had generated mature ascospores that were ejected onto the lid of the Petri dish. The ascospores were collected from the lid in sterile water using Pasteur pipettes and the number of ascospores was counted using a hemocytometer. Perithecia were manually collected and squashed between a microscope slide and a cover glass to break the perithecia open and release “rosettes” of developing ascospores. A Brightfield microscope was used to view the developing ascospores, which were photographed with a Canon Powershot A620 camera fitted with a microscope adaptor.

ALT levels in the sera from mice at 7 dpi were measured using a Cobas C111 analyzer (Roche) in the NUS comparative medicine in-house veterinary diagnostic laboratory. Histopathological analysis was performed as previously described by Costa et al. (10 (link)). Briefly, liver samples from humice were obtained at day 7 after DENV-2 inoculation. Afterward, samples were immediately fixed in 10% buffered formalin for 24 h and embedded in paraffin. Tissue sections (4 mm thick) were stained with hematoxylin and eosin (H&E) and evaluated under an Axioskop 40 microscope (Carl Zeiss, Göttingen, Germany) adapted to a digital camera (PowerShot A620; Canon, Tokyo, Japan). Histopathology scoring was carried out by a pathologist in a blinded fashion according to a set of custom-designed criteria described by Costa et al. (10 (link)). Hepatocyte swelling, degenerative changes, necrosis, and hemorrhage were each graded on a scale of 0 to 5 (0, absent; 1, minimal; 2, slight; 3, moderate; 4, marked; and 5, severe). The sum of the score was computed to derive the overall liver damage score. A total of two sections for each animal were examined.

Liver samples from euthanised mice were obtained at the indicated time points. After that, samples were immediately fixed in 10% neutral-buffered formalin for 24 hr and embedded in paraffin. Tissue sections (4 µm thicknesses) were stained with hematoxylin and eosin (H&E) and evaluated under a microscope Axioskop 40 (Carl Zeiss, Göttingen, Germany) adapted to a digital camera (PowerShot A620, Canon, Tokyo, Japan). Histopathology score was performed as previously described (Costa et al., 2012 (link)), evaluating hepatocyte swelling, degeneration, necrosis, and haemorrhage added to a five-point score (0, absent; 1, minimal; 2, slight; 3, moderate; 4, marked; and 5, severe) in each analysis. A total of two sections for each animal were examined, and results were plotted as the mean of damage values in each mouse.

For histochemical localization of GUS activity, leaf discs of potato and tomato were incubated with 0.5 mg/ml X-Gluc (5-bromo-4-chloro-3-indolyl b-D glucuronide, Biosynth AG) in 50 mM phosphate buffer (pH 7.0, 1 mM KFeCN and 0.05% (v/v) Triton-X100) overnight at 37 °C. The staining solution was removed and leaf discs were washed with 96% ethanol until tissue was cleared. The tissue was mounted on microscope slides in 50% (v/v) glycerol.
In order to detect H₂O₂ generation in detached plant leaves, DAB staining was performed. Leaf discs were placed in a solution of 1 mg/mL 3,3′-diaminobenzidine dissolved in 0.2 M PBS (phosphate buffer) and HCl was used to adjust the pH to 3.8. The leaf samples were placed overnight in light to optimize the staining reaction. The samples were cleared by boiling with 96% ethanol until the leaves were decolorized and transferred to fresh 70% ethanol for storage until microscopic examination.
Twelve preparations were observed for each genotype per time point with a bright field microscope (Zeiss, Germany). Pictures were taken with a Canon PowerShot A620 camera mounted on the microscope. The presence of H₂O₂ is visualized as a brown coloration.

Five flowers from six plants (n = 30) stage 15a [6 (link)] were dissected. Floral organs were analyzed and counted using a Zeiss Stemi-SV6 stereomicroscope. Photographs of flowers (10 days after anthesis) were taken using a Canon Power Shot-A620 camera and captured with Canon ZoomBrowserEX5.5.0.190. Petal size and cell number were determined as described in [33 ] using twenty flowers from four independent plants of each genotype grown under the same conditions.
For fluorescence-activated cell sorting (FACS) analysis, 2 young leaves, 50 flowers or 200 petals from 5 independent plants were used for each genotype. Nuclei isolation and FACS analysis were performed as described in [34 (link)] using MACSQuant VYB (Miltenyi Biotec) cytometer.
Statistical analyses were undertaken and graphics created for all measurements. Regression analyses and ANOVA using generalized linear models were performed using GenStat 15.1.0.8035. Graphs were created using Microsoft Excel 2010 and annotated in Adobe Photoshop 7.0.1.

To determine the linear growth rate of the mutant strains, 5 µL of conidia from a mutant isolate (5 × 10⁴ conidia in water) was spotted near the edge of a Petri dish containing Vogel’s 2% sucrose 2% agar medium and the mutants were grown at 30 °C. The extension of the hyphae across the agar medium was monitored by marking the location of the leading edge of the colony at 10 h and 20 h post-inoculation. The linear growth rate was calculated as the average hourly rate of extension of the colony leading edge in the time interval. These linear growth rate experiments were performed in triplicate and an average growth rate with a standard deviation was determined for the wildtype and mutant strains. To examine hyphal morphology, the growing edges of the colonies were viewed in a dissecting microscope and photographed with a Canon Powershot A620 camera fitted with a microscope adaptor.