Sem feg xl 30 microscope

Samples were collected and fixed with 4% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2–4 h at room temperature and then washed for 10 min, three times in the same buffer.
For TEM analyses, specimens were post fixed for 1 h in 1% osmic acid in cacodylate buffer. After dehydration in an ethanol series, samples were embedded in an Epon–Araldite 812 mixture and sectioned with a Reichert Ultracut S ultratome (Leica, Nussloch, Germany). Semithin sections (70 μm) were stained by crystal violet and basic fuchsin and then observed with a light microscope (Eclipse Nikon, Amsterdam, Netherlands); images were acquired with a Nikon DS-SM camera. Thin sections (70 nm) were stained by uranyl acetate and lead citrate and observed with a Jeol 1010 electron microscope (Jeol, Tokyo, Japan).
For SEM analysis, specimens were post-fixed for different times in a solution of 1% osmium tetroxide and 1.25% potassium ferrous-cyanide and dehydrated in an ethanol series and two times in hexamethyldisilazane. The samples were mounted on carbonated stubs and gold coated in an Emitech K250 sputter coater (Emitech, Baltimore, MD, USA) and observed with a SEM-FEG XL-30 microscope (Philips, Eindhoven, The Netherlands).

After rearing BSF larvae on PEd and PSd for 2 weeks, the substrates were collected to evaluate any structural alterations on the surface of the plastic particles (post-rearing samples). PEd and PSd kept in the same conditions without the larvae served as controls. All diet samples were stored at − 20 °C until use. Moreover, nonagarized PS and PE particles (i.e., plastic powders) were analyzed, too. After thawing, samples were placed in cell strainers (40-μm mesh size) (BD Biosciences, Milan, Italy) and extensively washed with 100 mM NaCl and Milli-Q water. Samples were then mounted on stubs, gold-coated with a Sputter K250 coater, and finally analyzed with a Scanning Electron Microscope (SEM)-FEG XL-30 microscope (Philips, Eindhoven, The Netherlands) (Centro Grandi Attrezzature, University of Insubria). For each condition, the analysis was performed in triplicate.

After incubation with 53D1 or Chi18H8, PM was fixed with 4% (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4, overnight at 4°C. After post-fixation with 1% (w/v) osmium tetroxide and 1.25% (w/v) potassium ferrocyanide for 1 h, samples were dehydrated in an ethanol series and then incubated in hexamethyldisilazane (two steps of 10 min each). Samples were mounted on stubs, carbon coated with a Sputter K250 coater, and finally observed with a SEM-FEG XL-30 microscope (Philips, Eindhoven, Netherlands).

To obtain three-dimensional imaging by SEM, tissues from leeches untreated or exposed to MWCNTs were fixed in 4% paraformaldehyde for 1 h at room temperature. The specimens, washed in PBS (pH 7.2), were dehydrated in an increasing series of ethanol, cleared in in xylene for 30 minutes and then penetrated with paraffin (melting point, 58–60°C, Bioptica, Milan, Italy), at 60°C over night. Paraffin sections (7 μm) were deparaffinized with xylene and dehydrated in an increasing series of ethanol. Slides were mounted on stubs, sputter coated with a thin layer of gold and then observed with a SEM-FEG XL-30 microscope (Philips, Eindhoven, The Netherlands).
To confirm the presence of aluminum, iron and cobalt associated to the crude MWCNTs powder and evaluate their entrance in leech tissues, samples were observed in backscattered electron mode with a scanning electron microscope coupled with an energy dispersive X-ray analyzer (EDAX Genesis 2000). Samples were stuck onto slide holders and sputter coated with a thin layer of gold. Photographic maps of element distribution obtained on the image frames were processed by Image Analysis (1994) (Soft-Image Software GmbH). These maps were then superimposed to each source image with Adobe Photoshop (Adobe Systems).

Adult aphids (6 in the wingless morph and 2 in the winged morph) were prepared as described by Sun et al. (2013) (link). Briefly, they were fixed in 70% ethanol for 2 h and cleaned in an ultrasonic bath for 1 min in the same solution. Finally, samples were dehydrated in 100% ethanol for 30 min, air-dried, coated in gold by K250 sputter coater (Emitech, Ashford, Kent, United Kingdom) and examined with SEM-FEG XL-30 microscope (Philips, Eindhoven, The Netherlands).

Reaching an OD600 of 0.6, E. coli bacteria were incubated as previously described. After treatments, cells were centrifuged for 10 min at 13,000 rpm, and bacterial pellets were fixed with Karnovsky fixative (2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate Buffer, pH 7.2) for 30 min at 4 °C. Samples were washed in 0.1 M cacodylate buffer (pH 7.2) and post-fixed in a solution of 1% osmium tetroxide and potassium ferrocyanide for 1 h. After several washings in PBS (pH 7.2) and dehydration with an increasing scale of ethanol, 20 µL of bacterial pellet resuspended in 100% ethanol were dried onto glass slides and finally subjected to critical point drying with hexamethyldisilazane. Images were acquired using SEM-FEG XL-30 microscope (Philips, Eindhoven, The Netherlands).

After rearing BSF larvae on PEd and PSd for 2 weeks, the substrates were collected to evaluate any structural alterations on the surface of the plastic particles (post-rearing samples). PEd and PSd kept in the same conditions without the larvae served as controls. All diet samples were stored at − 20 °C until use. Moreover, nonagarized PS and PE particles (i.e., plastic powders) were analyzed, too. After thawing, samples were placed in cell strainers (40-μm mesh size) (BD Biosciences, Milan, Italy) and extensively washed with 100 mM NaCl and Milli-Q water. Samples were then mounted on stubs, gold-coated with a Sputter K250 coater, and finally analyzed with a Scanning Electron Microscope (SEM)-FEG XL-30 microscope (Philips, Eindhoven, The Netherlands) (Centro Grandi Attrezzature, University of Insubria). For each condition, the analysis was performed in triplicate.