K850 critical point dryer

The stamen primordia and gynoecium primordia were dissected carefully and photographed using a LEICA S6D stereomicroscope (Leica Microsystems, Wetzlar, Germany). Then, the floral buds, stamen primordia, and gynoecium primordia were fixed in FAA for 24 h for SEM and paraffin sectioning. Some fixed materials were dehydrated in an ethanol series for 20 min per step and dried using CO₂ as the exchange agent by an EMITECH K850 critical point dryer (Emitech, Canterbury, British). Then, the samples were coated with gold by an Edwards E-1010 ion sputter golden coater (Hitachi, Tokyo, Japan) and photographed with an FEI Quanta 200 scanning electron microscope (FEI, Eindhoven, Netherlands).
The remaining fixed materials were dehydrated in an ethanol series, transparentized in a xylene series, infiltrated in paraffin, embedded in paraffin wax, and sectioned at an 8 µm thickness with a LEICA RM2145 rotary microtome (Leica Microsystems, Wetzlar, Germany). Finally, the sections were stained with safranin and fast green and photographed with a Zeiss AXIO Axioscope A1 fluorescence microscope (Carl Zeiss, Jena, Germany).

The morphology of MG-63 osteoblasts was analyzed by field emission scanning electron microscopy (FE-SEM, 5 kV; Merlin VP compact, Carl Zeiss, Oberkochen, Germany). The cells were fixed with 2.5% glutaraldehyde (Merck), dehydrated through a grade series of ethanol (30%, 50%, 75%, 90%, 100%), and dried in K850 critical point dryer (Emitech, Taunusstein, Germany). As the final step, cells were sputtered with a 20 nm gold-layer (SCD 004, BAL-TEC, Balzers, Liechtenstein, Liechtenstein). To image the cells, a high efficiency secondary electron detector (HE-SE) was used.
The cell spreading was analyzed by using ImageJ (Version 1.51f, Wayne Rasband, National Institutes of Health, Bethesda, MD, USA) of the FS-SEM images. For this, 40 cell areas per sample were calculated, and the statistical analyses were performed with GraphPad Prism (Version 7.02, GraphPad Software Inc., La Jolla, CA, USA) by non-parametric Kruskal–Wallis test post hoc uncorrected Dunn’s test (analysis of variance) (* p < 0.001). The data were presented as mean ± standard error of the mean (s.e.m.).

Images of mature leaves were taken with a Nikon 7100 camera (Nikon, Japan). Fresh leaves were first fixed in FAA (methanol: acetic acid: ethanol: water = 10:5:50:35). Next, small leaf pieces were dehydrated in an increasing alcohol series and isoamyl acetate series, and then, critical-point dried in CO₂ with a K850 critical-point dryer (EMITECH, Ashford, England). Leaf pieces were then mounted on stubs and sputter coated with gold–palladium using a JS-1600 sputter coater (HTCY, China). The materials were photographed with a Hitachi S-3400 scanning electron microscope (SEM, Hitachi, Japan) at 15 kV.

The cestodes were collected carefully from the intestines of small ruminants (sheep/goats) and identified after staining, using keys [21 –24 ]. A carmine stain was on mature proglottids. Proglottids were fixed and washed in 70% ethanol. Staining was with iron hydrochloric carmine, destained in acid ethanol (100 mL 70% ethanol + 2 mL concentrated HCl), dehydrated in a graded ethanol series, cleared with eugenol (clove oil), and mounted in Canada balsam. Stained specimens were examined and photographed under a Leitz photo research microscope. To confirm detailed surface morphology, SEM observations were performed. For SEM, worms were isolated carefully from intestines and placed into a small amount of saline buffer. For living tapeworms, scolex, mature, and gravid proglottids of many specimens of each species were fixed overnight in cold 2.5% glutaraldehyde in a 0.1 M sodium cacodylate buffer at pH 7.4. Then, they were dehydrated in a graded ethanol series and dried using CO₂ in an Emitech K850 critical point dryer. After being mounted on metal stubs, specimens were coated with gold/palladium in a Quorum Technologies SC7640 sputter coater and examined with a Hitachi S-3400N scanning electron microscope at acceleration voltages between 3 and 20 kV in the “Service d'Etude et de Recherche en Microscopie Electronique de l'Université de Corse.”

The microstructure of the ant nests was examined and recorded using an Olympus DP21 digital camera mounted on an Olympus BX63 compound microscope or VEGA3 TESCAN SEM (Figure 1B–J). Fresh samples in the field were recorded using Panasonic DMC-TZ60 digital camera (Figure 1A1). For the SEM techniques, samples were dried at the critical point of CO2 using an Emitech K850 Critical Point Dryer. The dried samples of carton were coated with a layer of gold using an Emitech K550X Sputter Coater. Thereafter, the samples were placed directly in the SEM chamber for examination.

For observations of the epidermis surface, small pieces of ray florets (about 3 × 3 mm) were fixed in a 2.5% or 4% glutaraldehyde solution in 0.1 M phosphate buffer (pH 7.0) at room temperature for 2 h and then washed in phosphate buffer four times at 20-min intervals. The fixed plant material was dehydrated in graded ethanol series and immersed in absolute ethanol (POCH, Gliwice, Poland) three times for 30 min. Next, the floret samples were critical-point dried in liquid CO₂ using a K 850 Critical Point Dryer and sputter-coated with gold (20 mm thickness) using K 550X (Emitech, Ashford, UK). The observations were carried out using a Tescan Vega II LMU scanning electron microscope (Tescan, Brno, Czech Republic) at an accelerating voltage of 30 kV [69 (link)].