Em cpd030

Parasite processing was carried out using glass coverslips pre-coated with 1 mg/ml poly-L-lysine. Cells were fixed for 1 h in 2.5% glutaraldehyde diluted in cacodylate buffer [0.1 M (pH 7.2)] and then were adhered to coverslips, post-fixed for 1 h with 1% osmium tetroxide diluted in cacodylate buffer. Samples were dehydrated in a graded alcohol series (50%, 70%, 90%, and two exchanges of 100% ethanol for 10 min each step) and then were critical-point dried in a Leica EM CPD030 apparatus (Leica, Wetzlar, Germany). Specimens were coated with platinum in a Leica EM SCD050 before visualization using a Zeiss EVO 40 VP scanning electron microscope. Measurements of cells lengths were made using the program AxioVision4 and were based upon the SEM images. Statistics were calculated using the Wilcoxon-Mann-Whitney test in GraphPad Prism 6 software (GraphPad Software). P values less than 0.05 were considered statistically significant.

Adult worms collected by perfusion were immediately transferred to supplemented RPMI medium; parasites were distributed in 6-well plates (adults: 10 paired worm couples per well) with medium. The worms were kept in culture (oven at 37°C and 5% CO₂) for 2 h for adaptation, and then NJ series compounds or the equivalent amount of 0.1% DMSO vehicle were added.
Ultrastructural analysis was performed with scanning electron microscopy. Adult worms incubated at different concentrations of NJ series compounds or with 0.1% DMSO vehicle for 1 and 2 days were fixed with modified Karnovsky reagent (1% paraformaldehyde, 2.5% glutaraldehyde, 1 mM calcium chloride in 0.1 M sodium cacodylate buffer, pH 7.4) and after the fixing stage the material was washed with sodium cacodylate buffer (0.1 mol / L, pH 7.2) and post-fixed with 1% osmium tetroxide (w / v) for 1 h.
Samples were dehydrated with increasing concentrations of ethanol and then dried with liquid CO₂ in a critical-point dryer machine (model Leica EM CPD030, Leica Microsystems, Illinois, USA). Treated specimens were mounted on aluminum microscopy stubs and coated with gold particles using an ion-sputtering apparatus (model Leica EM SCD050, Leica Microsystems, Illinois, USA) [34 (link)]. Specimens were then observed and photographed using an electron microscope (FEI QUANTA 250, Thermo Fisher Scientific, Oregon, USA).

Scanning electron microscopy (SEM) (JSM-5900LV, JEOL USA, Inc., Peabody, MA, USA) was used to characterize the thickness of the coatings and to identify the coating morphology changes after coating incubation in a culture medium with or without cells. The effects from both cell types were evaluated by SEM. The MC3T3-E1 or RAW 264.7 cells were cultured on disks coated with AntiA-bCaP-FGF2 or AntiA-bCaP-PEM30-FGF2. After LIVE^® staining was performed, the cells were removed by incubation in Trypsin-EDTA (Sigma, St. Louis, MO, USA) and were washed three times with ultrapure water (reverse osmosis), and dried with a series of ethanol solutions for SEM imaging. The disks with PEM coatings were critically point dried after ethanol dehydration (LEICA EM CPD030, Leica Microsystems Inc., Buffalo Grove, IL, USA) to preserve the delicate surface structure of the PEM film. For consistency, the bCaP only coated disks were also critically point dried. The disks were sputtered coated (DESK V, Denton Vacuum, LLC, Manchester, NJ, USA) and imaged using a TM-1000 SEM (Hitachi High-Technologies Corporation, Tokyo, Japan). The SEM analysis was conducted on five areas per quadrant, and on at least three samples per group.

The confrontation of C. rosea and B. cinerea was also monitored by SEM. The agar strips (2 × 0.5 cm) were cut from the edges of C. rosea and B. cinerea colonies and placed 3 cm from each other on the surface of 5 mL PDA in a 9 cm Petri dish. The plates were sealed with Parafilm, and the fungi were co-cultured at 26 °C for 6 days until G67-1 strain overgrew B. cinerea colonies. The conjoint regions containing both fungi were cut into ~5 mm blocks, fixed in 3% glutaraldehyde at 25 °C in the dark for 48 h, and stored at 4 °C in a refrigerator before observation. Three replicates were included for each sample. The specimens were gently dried using a Leica EM CPD030 instrument (Leica Microsystems, Australia) and coated with gold powder. Interactions between the mycoparasite and its host were detected under a Hitachi SU8010 scanning electron microscope (Hitachi High-Technologies Co., Tokyo, Japan) with an accelerating voltage of 10 kV.

To study the morphological characteristics of the pathogen, the 7-day-old fungal culture of each isolate was observed under a light microscope Olympus BX61. The data were recorded using CellSens Dimension (Olympus) software. The color and descriptions of conidia and mycelium were made by using the color chart of Rayner and the standard identification manual (Rayner, 1970 ; Simmons, 2007 ). The shape and size of 30 conidia per-isolate were observed and recorded. Furthermore, SEM analysis was carried out for ultrastructural observation. For sample preparation, the fungal mycelia were rinsed twice with phosphate buffer (pH 7) and fixed with 2.5% glutaraldehyde buffer (pH 7) for 24 h. Then, the sample was rinsed three times with 10 mM phosphate buffer and fixed for 2 h in 1% osmium tetroxide. Subsequently, each sample was subjected to gradient dehydration with 30, 50, 60, 70, 80, 90, 95, and 100% ethanol solution sequentially for 15 min. The samples were dried on a Leica EM CPD030 automated dryer (Leica Microsystems Inc., Germany) and fixed by a carbon tape on the sample stage of SEM. The gold coating of samples was done by ion sputtering “HITACHI MC1000.” The morphology was observed using a HITACHI SU8010 scanning electron microscope (Hitachi High-Technologies Corporation. Tokyo, Japan).

Different states of microspheres (1 g) were prepared and cleaned twice in water. After cleaning, 3 mL ethanol in water (7:3 v/v) was added and the microspheres were fixed for 24 h. After this, samples were gradient-eluted in 70%, 80%, 90%, and 100% ethanol, each for 10 min. The samples were then dried on a Leica EM CPD030 automated critical point dryer (Leica Microsystems Inc, Wetzlar, Germany). Ultrathin sample sections were prepared using an ultramicrotome (Leica EM UC6) (Leica Microsystems Inc, Wetzlar, Germany). The surface morphology was observed using a HITACHI SU8010 scanning electron microscope (Hitachi High-Technologies Corporation, Tokyo, Japan).