Em uc6 microtome

For light microscopy, larvae were fixed in glass vials with 2.5% glutaraldehyde, 2% paraformaldehyde (PFA) and 0.1% Sorenson’s phosphate buffer (pH 7.3) and placed at 4°C overnight. Samples were transferred to 1% osmium tetroxide before an ethanol gradient dehydration. Larvae were embedded in agar epoxy resin and sectioned using a glass knife and a Leica EM UC6 microtome. Retinal sections were placed on glass slides and stained with Toluidine blue (Sigma Aldrich, UK) and imaged using a Leica DMLB bright field illumination microscope with a Leica DFC 480 camera. For transmission electron microscopy (TEM), sections from light microscopy were stained with uranyl acetate and lead citrate and imaged with FEI Tecnai 120 transmission electron microscope (Thermo Fisher Scientific, Waltham, MA, United States). Morphological analysis was performed on retinal sections for light microscopy and using arbitrary lines from OLYMPUS cellSens Standard software with SZX2-ILLT Olympus stereo microscope. First, a straight arbitrary line connecting both marginal zones named here front plane was traced. Axial length was measured using a perpendicular line (named optic axis here) to front plane from anterior lens surface to RPE. Retinal layers were all measured at the optic axis. Measurements were done using an Olympus SDF PLAPO 1.6XPF objective and 11.5X magnification.

HMVECs were treated by AuNPs of three different sizes for 30 min and then washed twice by PBS. The cells were fixed by the mixture of 2.5% glutaraldehyde and 0.1 m sodium cacodylate overnight at 4°C, followed by osmium tetroxide polymerization (2%, 30 min at 20°C), uranyl acetate staining (1% overnight), and resin embedding (24 h, 60°C). Thick slices of 70 nm were prepared using a histo diamond knife (DiATOME, Switzerland) and a Leica EM UC6 microtome. The slices were placed on carbon‐coated copper grids (100 mesh, formvar film, ProSciTech), and images captured using a HITACHI HT7700 transmission electron microscopy operated at 80 kV.

Dissected ovaries were rinsed in locust Ringer solution. Images of the ovaries and ovarioles were obtained using a light microscope (Zeiss SteREO Discovery.V8) equipped with an AxioCam ICc3 camera using the AxioVision 4.7 (Carl Zeiss-Benelux). Oocyte sections were made according to Billen^{41 (link)}. In short, the ovarioles were fixed in 2% glutaraldehyde in sodium cacodylate buffer and postfixed in 2% osmium tetroxide in the same buffer. Afterwards, samples were dehydrated in a graded acetone series and embedded in araldite. Semi-thin (1 µm) sections (Leica EM UC6 microtome) were stained with methylene blue and thionin. Images of the oocyte sections were obtained with a light microscope (Zeiss Axio Imager Z1) equipped with an AxioCam MRm camera (1388 × 1040 pixels) using the Imaging software program Zen 2012 (Blue Edition; Carl Zeiss-Benelux).

Nauplii were fixed for 12 h in 2.5% glutaraldehyde prepared in phosphate-buffered saline (PBS). Thereafter, they were washed and post-fixed in 1% osmium tetroxide, dehydrated in a graded acetone series, and embedded in Spurr resin. Sections of 70 nm in thickness were obtained with a Leica EM UC6 microtome (Wetzlar, Germany), stained with 2% uranyl acetate and Reynold's solution (0.2% sodium citrate and 0.2% lead nitrate), viewed by a JEM-1230 transmission electron microscope (JEOL, Akishima, Tokyo, Japan), and photographed at a voltage of 70 kV.

From one alate gyne, one dealate queen and four workers, the middle part of the body was separated by making a transverse cut at the level of the midlegs anteriorly and behind the first gastral segment posteriorly. The tissues were fixed in cold 2% glutaraldehyde, buffered at pH 7.3 with 50 mM Na-cacodylate and 150 mM saccharose. After postfixation in 2% osmium tetroxide in the same buffer, tissues were dehydrated in a graded acetone series and embedded in Araldite. Serial longitudinal semithin sections with a thickness of 1 μm were made with a Leica EM UC6 microtome and stained with methylene blue and thionin. The sections were observed and photographed with an Olympus BX-51 microscope. For each individual, we measured the length of the stridulation file and distance between ridges and counted the number of ridges.

The antennae used for Transmission electron microscopy (TEM) were excised and prefixed for 2 days with paraformaldehyde (4%) and glutaraldehyde (2.5%) in 0.1 M phosphate buffered saline (PBS, pH 7.2), then postfixed for 1 h with 1% OsO₄ in 0.1 M PBS (pH 7.2), and followed by dehydration in an ethanol series solutions (30, 50, 70, 80, 90, 95–100%) for 3 min each. After being dehydrated with pure acetone three times for 10 min each, the samples were embedded in Epoxide resin 618 through mixtures of 2: 1, 1: 1, 1: 2 of acetone and Epoxide resin 618 (Serva, Heidelberg, Germany) and then kept in pure Epoxide resin 618 overnight. Polymerization was accomplished with heating from 30 to 60°C (5°C/6 h), at 60°C for 48 h in tightly closed gelatin capsules filled completely with the resin monomer. Ultrathin sections were cut with a diamond knife (Diatome, Bienne, Switzerland) on a Leica EM UC6 microtome (Wetzlar, Germany) and then mounted on Formvar-coated grids. The sections were observed on a HITACHI H-7500 TEM (Hitachi, Tokyo, Japan). Pictures were only adjusted for brightness and contrast.

Treated or untreated U-937 cells were fixed with 2.5% glutaraldehyde (4 °C, overnight). After washing with PBS three times, cells were incubated with 1% osmium tetroxide for 1 h on a rotator for the post-fixation step. After three washing steps cells were resuspended in 6% gelatin for 10 min and pelleted at 3000 rpm. Excess of gelatin was removed and the cells were submitted to a new fixation step in 2.5% glutaraldehyde for another 10 min. The samples were washed three times with distilled water before being cut into 1mm³ cubes for further processing. Cubes dehydration was achieved using solutions of increasing ethanol concentrations (from 25 to 100% alcohol and then 100% acetone) before being progressively permeated with Epon-Araldite (from 1:6 ratio of Epon-Araldite: acetone to 100% Epon-Araldite). Three Epon-Araldite baths were made on the following day (40–50 °C). Polymerization was performed (60 °C for 24 h). Sample’s Sects. (90-100 nm) were prepared and collected on slot copper grids using a Leica EM UC6 microtome. Counter-staining was performed using uranyl acetate and lead citrate and then observed using a Philips transmission electron microscope JEOL JEM-1010 (100 keV electron beam energy). Pictures acquisition was done with a Gatan ES500W Erlangshen camera (1350 × 1040 pixels).