Application suite software

Specimens are deposited in the following collections (abbreviation and curator in parentheses): Instituto Butantan, São Paulo (IBSP, A.D. Brescovit) and Museu Paraense Emílio Goeldi, Belém (MPEG, A.B. Bonaldo).
Morphological terms follow Brescovit et al. (2018) (link), except for macrosetae of endites which follow Baert (2014) . Descriptions and measurements were performed using a Leica 165C stereomicroscope, while photographs were taken with a Leica DFC 500 digital camera mounted on a Leica MZ16A stereomicroscope. Focal range images were made using Leica Application Suite software, version 2.5.0. Total and femur lengths were measured in lateral view without detaching any part from the specimen. All measurements are in millimeters. Female genitalia were excised with a sharp needle and photographed mounted on Hoyer´s microscope slides. For scanning electron microscopy (SEM), body parts were dehydrated in a series of graded ethanol washes (80% to 100%), critical point dried, mounted on metal stubs using adhesive copper tape and nail polish for fixation and covered with gold. SEM images were taken with FEI Quanta 250 and LEO 1450VP scanning electron microscopes, at Laboratório de Biologia Celular of Instituto Butantan, São Paulo and Museu Paraense Emílio Goeldi, Belém, respectively.

The MMVECs (2 × 10⁴ cells per well) were seeded in 96-well plates which have been filled with 50 μl matrigel and solidified in 37°C. Cells were cultured in CM supplied with 2.5% FBS for 6 hours. To observe the formation of tube-like structures, five optical fields (10× magnification) per well were randomly chosen and analyzed by a LEICA DMI 4000B microscope with Leica Application Suite software.

Larvae were cultured in Danieau’s medium and scored daily up to 10 days post fertilization (dpf) for any morphological or behavioral abnormalities (e.g., pericardial edema, head, eye, or jaw malformations, bent spine, and lack of touch response) and lethality. The larvae were photographed using a stereomicroscope (Leica MZ10 F) equipped with a digital camera (DFC310 FX) and Leica Application Suite software (version 3.6.0). Dead larvae were immediately collected for genotyping as described before.

Anthers were fixed in FAA (10% formaldehyde, 3% acetic acid and 43.5% ethanol) placed under vacuum for 1 h and then keep room temperature. After dehydration in a graded ethanol series and diaphaneity in clearing medium, the material was embedded in Paraffins (from HuaShenPai). Sections (6 μm) were obtained with a Leica Reichert Supernova microtome, placed on glass slides, and stained with a solution of 1%(w/v) toluidine blue O (toluidine blue O 1 g, 95% alcohol 4 mL, 10% acetic acid 10 mL, distilled water 86 mL). Sections examined using a Leica fluorescent compound microscope and Images were captured with a Leica DFC420 camera, and processed with Leica Application Suite software.
The images of whole morphology of WT and mutant were captured using SONY DSC-H50 camera. And the flowers, siliques and seeds morphology were examined using a Leica dissecting microscope.

To measure the autofluorescence of tomato plastids, three 7, 17 and 17 DPA fruit of M82, AtOR^WT and AtOR^His lines were harvested. A piece of equatorial pericarp was cut into thin slices. The autofluorescences of chlorophylls and carotenoids were monitored under Leica TCS SP5 Laser Scanning Confocal Microscope, using the setting described by D'Andrea et al. (2014). The 488 nm light was used as excitation source. Chlorophyll fluorescence was taken at 650–700 nm, and carotenoid fluorescence was taken at 500–550 nm.
To measure plastid sizes, plastid pictures in cells of 7 DPA fruit from M82, AtOR^WT 20 and AtOR^His 23a T1 transgenic plants were taken under Confocal Microscope with the settings as above. The diameters of plastids were measured by the scale tool of the Leica Application Suite software.

Fiber diameters were determined by reflected light microscopy using a DM4000 M instrument (Leica Microsystems, Wetzlar, Germany) at 100–200× magnification, and images were captured using Leica Application Suite software. Ten images representing different areas of each non-woven fiber were used to determine the average fiber diameter. DSC was performed on all melt-electrospun fibers to determine the effect of additives and the electrospinning process. DSC was also carried out on the PFs and DFs, as well as the melt-electrospun PLA fibers under the same testing conditions. The thermal transition temperatures and X_c values were compared. Polarized optical microscopy (POM) was used to investigate the crystallinity of the melt-electrospun filaments, PFs and DFs. An Olympus BX53 microscope and DP26 camera (Olympus BV, Leiderdorp, The Netherlands) were used to capture the images at 50× magnification. The images were screened for birefringence. The relationship between the electrical resistance, melt viscosity, and average fiber diameter was visualized in surface plots using Minitab19 analysis software.