Evo40 scanning electron microscope

Selected samples from the roots, stems and fully expanded leaves near the primary veins were excised, sectioned into small pieces and frozen in liquid nitrogen. The samples were then freeze-dried and coated with gold for 60 s (ca. 1 nm thickness of gold) by a Sputter Coater (Cressington model 108, Ted Pella Inc., U.S.), and then detected by a scanning electron microscope (SEM; SU8010, Hitachi, Japan). The cross sections were viewed at an accelerating potential of 20 kV under high vacuum mode with backscatter detection. Images were captured at different magnifications. For each species, at least three plants were examined for each treatment group. Digital photographs were taken using an EVO 40 scanning electron microscope (Zeiss).

B16F10 cells were grown on sterile 5 mm cover slips in 24 well plates. After treatments, the cells on the cover slips were washed with PBS and fixed with 2.5% glutaraldehyde solution containing 4.5% glucose buffered with 75 mM cacodylate buffer (pH 7.2) for 3 h at room temperature. After 3 washes with 100 mM cacodylate buffer, secondary fixation was achieved by the addition of 1% osmium tetroxide buffered with 50 mM cacodylate (pH 7.2) for 3 h at room temperature. Subsequently, the cells were washed with distilled water, dehydrated in an ethanol series (25%, 50%, 75%, 90% and 100%) and then a 1∶1 mixture of ethanol/acetone, and subsequently kept in pure acetone. Finally, critical point drying was performed with CO₂ in an E 3000 critical point drying apparatus (Quorum Technologies, Newhaven). The specimens were mounted on adhesive carbon discs and sputter-coated with gold in a SC7620 Sputter Coater (Quorum Technologies, Newhaven), and images were taken with an EVO40 scanning electron microscope (Carl Zeiss GmbH, Oberkochen) at 20.0 kV. Quantitation of the image surfaces was carried out by using ImageJ. Cell borders were drawn and pixel numbers within the borders were obtained and converted to µm² units. For each sample, measurements were made on 30 cells, and t-tests were then performed.

SEM images were generated using a Zeiss EVO 40 scanning electron microscope at an acceleration voltage 10.00 kV. Samples were prepared by pipetting a drop of liposomes suspension onto the surface of the Thermanox^® plastic coverslips (ThermoFisher Scientific, Fremont, CA, USA), fixed with 2.5% glutaraldehyde, incubated for 1 h for sedimentation, and dehydrated in alcohols of increasing concentrations and acetone.
Thereafter, the samples were completely dried in carbon dioxide according to the critical-point drying method by using a BALTEC 030 (BAL-TEC AG, Balzers, Liechtenstein). The samples were then placed on the surfaces of aluminium substrates and were coated with chromium.

Fresh, hand-made sections were used for the histochemical analyses. The distribution of phenols within trichomes of C. avellana was examined using toluidine blue [31 ]. Each section was observed at 10, 20 and 40 × magnification with an Axioskop 20 microscope. The proportion of trichomes with phenolic compounds staining, relative to the total number of observed trichomes, was then calculated.
For anatomical measurements, 1-mm² sections of C. avellana leaves were fixed with a mixture of 4% (v/v) glutaraldehyde and 4% paraformaldehyde (1:1; w/v; pH 6.8; Polysciences, Warrington, USA) in 0.5 M cacodylate buffer at pH 7.2 for 24 h. The specimens were then washed in distilled water, dehydrated in a graded ethanol series followed by a graded acetone series, and dried in a critical point dryer (Balzers CPD-030) using CO₂ as a transition fluid. Dried sections were mounted on clean aluminum stubs with double-sided adhesive graphite tabs. Mounted specimens were coated with gold (12–15 nm thick), using a Balzers SPD-050 sputter coater. Sections were examined on an EVO40 scanning electron microscope (Carl Zeiss, Germany) and digitally photographed.

P. multocida strains were grown on glass coverslips in RPMI 1640 medium without phenol red or glutamine (Lonza, Walkersville, MD) and incubated at 37°C under stationary conditions for 48 h. The coverslips were gently washed and fixed in a solution of 5% glutaraldehyde, 4.4% formaldehyde, and 2.75% picric acid in 0.05% sodium cacodylate buffer for at least 1 h. Sequential dehydration of the sample was carried out with 25, 50, 70, 80, and 95% ethanol. SEM was performed as previously described (62 (link)), with a Carl Zeiss, Inc., EVO40 scanning electron microscope.