Stemi 2000 c microscope

A light microscope (LM) (AxioCam ICc 3 on a Stemi 2000-C microscope, Carl Zeiss AG, Oberkochen, Germany) was used for fracture surface analysis to measure the average crack length of prematurely removed CT specimens after manual cryofracture (see also Section 4.1). A scanning electron microscope (SEM) (EVO MA10 Carl Zeiss Microscopy GmbH, Jena, Germany) with an acceleration voltage of 10 kV was primarily used to visualize the post-failure surface structures depending on the environmental medium. For this purpose, the specimens were loaded in the tensile creep device until plastic deformation was reached, the residual cross-section was then cut with a razor blade to enable exemplification of the top view in the SEM. The SEM was used to visualize the fracture surface structures in more detail with magnification levels up to 1000×. Specimens were gold sputtered in a SCD 050 sputter coater (Leica Microsystems, formerly Bal-Tec Balzers, Wetzlar, Germany) for 100 s at a current of 40 mA at room temperature reaching a layer thickness of approx. 15 nm.

Fruits were evaluated in drnl-2 and Ler plants, which were germinated and grown under the same conditions as in the rest of the experiments. The numbers of fruits (siliques) and pistils that did not develop into fruits per plant were registered (n = 14 plants). Fruits were collected and classified according to their phenotype (n = 205 fruits). For the phenotypic analysis of pistils that did not develop into fruits, 199 pistils present along inflorescence stems were analyzed. Images were captured using a Stemi 2000-C microscope (Carl Zeiss, Oberkochen, Germany).

Digital images of colonies were collected using a Zeiss Stemi 2000-C microscope equipped with an Infinity 2 digital camera and Infinity Analyzer software (Lumenera Corporation, Ottawa, Canada). Differential interference contrast (DIC) images of cells were captured using a Zeiss Inverted Microscope (Axio Observer) fitted with an AxioCam HR. Images were processed with AxioVision Rel. 4.8 (Zeiss, Germany). To compare cellular phenotypes, eight or more images containing >250 cells were analyzed using CellProfiler v2.1.1 (Broad Institute of MIT and Harvard, Cambridge, MA) for each phenotype. Images were processed in the following manner: Edges were enhanced using the Sobel method, then a threshold was applied and cells identified using the mixture of Gaussian method. The eccentricity, form factor, and ratio of maximum and minimum Feret diameters of each cell were then calculated. Average and standard error were calculated using Microsoft Excel.

The effect of monacolin X on angiogenesis was evaluated using the CAM assay, following a method described previously with minor modifications.^{16 (link)} Fertilized chicken eggs were kept in a humidified egg incubator at 37 °C. The eggs were positioned horizontally and rotated several times. After 4 days of incubation, a 1 cm² window was carefully created on the broad side of the egg to assess the extent of embryonic blood vessels. The normal development was verified, and embryos with malformations or dead embryos were excluded. Then, about 2 mL of albumen was aspirated from each egg through the small window. After removal of albumen, monacolin X (15 μM, 30 μM and 60 μM) on a small disc filter paper was directly placed on the small window created before. At least five eggs were used for each dose. The control group was treated with albumen (100 μL per egg), while positive control group was treated with 20 ng mL⁻¹ of VEGF. After treatment, each egg was observed under a Zeiss Stemi 2000-c microscope equipped with Axiocam MRc 5 Zeiss, and blood vessels were photographed. The antiangiogenic effects of monacolin X on the CAMs were quantified Mean vessel area as a percentage of the total area, mean vessel length and mean the number of branch points which were marked using Wimcam software.^{19 (link)}

Ticks were morphologically identified to the species level using standard taxonomic keys (Walker et al., 2003 ) by light microscopy using a Stemi 2000-C microscope (Zeiss, Oberkochen, Germany). In addition, representative individual leg samples of morphologically identified ticks were used for DNA isolation followed by PCR-based molecular identification of tick samples through partial sequencing of the cytochrome oxidase subunit I (COI), 16S ribosomal (r)RNA, and 12S rRNA genes. We performed PCR in 10-µL reaction volumes consisting of 2 µL 5× HOT FIREPol® Blend Master Mix (Solis BioDyne, Tartu, Estonia), 0.5 µL of 10 µM forward and reverse primers (Table 1) and ~25 ng DNA template in a ProFlex PCR systems thermocycler (Applied Biosystems, Foster City, CA, USA). The amplification conditions consisted of an initial denaturation at 95°C for 15 min, 35 cycles of denaturation at 95°C for 20 s, annealing at 55°C (for both CO1 and 16S rRNA) and 50°C (for 12 rRNA) for 30s, and extension at 72°C for 30 sec, and a final extension of at 72°C for 7 min.

Modified phloem sandwiches (Bedard, 1933 (link)) were used to study maternal gallery construction and egg laying behavior. In short, a 15 × 8 cm strip of phloem was peeled from a freshly cut P. abies log and placed between two 18 × 10 cm Plexiglas panels. The panels were sealed together with electrical insulation tape and secured with four binder clips. A male bark beetle was placed in an opening in the top panel and covered with a piece of plastic mesh. After 24 h, a female beetle was added if the male had initiated a mating chamber. Twelve successfully mated pairs were observed with an EOS 600D (Canon) camera mounted on a Stemi 2000-C microscope (Zeiss). The different life stages were photographed and egg laying behavior was recorded whenever possible.

A Zeiss Stemi 2000-C microscope was used to analyze the lipids, ROS accumulation and GUS signal, the images were saved as jpg files using an Axi-Vision Camera. Live-cell microscopy was performed on a Zeiss 700 confocal laser scanning microscope using a 488 nm emission filter to detect the GFP signal.