Leo 912ab

Morphology of PLGA/PEI NPs was examined by field emission scanning electron microscopy (SEM) (FE-SEM, Mira IIIFEG, TESCAN-UK, Ltd). The sample solution in deionized water (1 mg/ml) was dehydrated on a metal stub for FE-SEM analysis. The morphology and bilayer configuration of PLGA/PEI co-polymer were also investigated by transmission electron microscopy (TEM) (Leo 912 AB, Carl Zeiss, Germany).

To investigate the morphology of CUR-NP and to confirm size data obtained by DLS measurements, AFM was performed using a Nanowizard 3® (JPK Instruments, Berlin, Germany) and a Digital Nanoscope IV Bioscope (Veeco Instruments, Santa Barbara, CA, USA), respectively. Formulations were diluted 1:100 with water and 20 µL of the diluted sample were placed onto a silica wafer or an untreated microscopic glass slide. The samples were left to settle onto the surface for a few minutes and the remaining fluid was absorbed by a lint-free wipe (Kimtech Precision Wipes, Kimberly-Clark, Fullerton, CA, USA). Measurements were performed in tapping mode, in which the cantilever oscillated with determined amplitude close to its resonance frequency, with scan rates from 0.5 to 1 Hz. A HQ:NSC16/AL_BS (Anfatec Instruments, Oelsnitz, Germany) cantilever was used [37 (link)]. Data were processed using JPKSPM data processing software (v. 5.1.8, JPK instruments).
For the TEM analysis, the nanoparticle suspension was applied onto 300-mesh copper grids. Samples were then negative stained thrice with 2% uranyl acetate (pH 4.2), which was alternated by washing steps with water. The samples were then allowed to dry overnight before being examined under the TEM (LEO 912 AB, Carl Zeiss, Jena, Germany) [22 (link)].

Following the RPM run, the spheroids derived from passage 3 chondrocytes were fished from the medium after 7 d, 14 d, 21 d and 28 d cultivation and fixed in Karnovsky fixative. The samples were then embedded in EMbed-812 resin blocks using a LYNX EI tissue processor (Leica Biosystems, Nussloch, Germany). After a first assessment of semi-thin sections (0.8 µm, toluidine blue/fuchsin double staining) by light microscopy, representative blocks were chosen for further ultra-thin sectioning (80 nm). Contrasting was done with Pb-citrate and U-acetate. TEM analyses were performed with a LEO912AB (Zeiss, Oberkochen, Germany) instrument, equipped with a 2k × 2k pixel side-entry CCD-camera (TRS, Moorenweis, Germany) and the iTEM image analysis software (OSIS, Münster, Germany).

ZnO/CeO₂ NCs were studied by PXRD (Advance-Bruker, Germany), in which the sample in powder form was analyzed from 2theta values in the range of 10–70° using Cu-radiation. The nanocomposites were scrutinized using FTIR spectroscopy by a Shimadzu8400 device, where KBr pellets of the samples were made and FTIR was set from the 400 to 4000 cm⁻¹ region. TEM was performed using a ZEISS LEO 912 AB. The sample was dispersed in water using ultrasound waves, then one drop of the suspension was poured onto a copper plate and dried and used for TEM analysis. For FESEM, a TESCAN device (MIRA 3) was used, and the sample was in powder form. DLS was performed using a Particle Size Analyzer, along with Vasco3 and Zeta potential. For DLS and Zeta potential analyses, 2 mg of the sample were dispersed using ultrasound waves in 10 mL of distilled water at a pH of 7.

Isolated ovaries (n = 4, from 2 females per group) were fixed in 4% glutaraldehyde in sodium cacodylate buffer (0,1 M, pH 7.4) for 2 h and postfixed with 1% osmium tetroxide in the same buffer for 2h. After dehydration in a graded ethanol series, the specimens were placed in propylene oxide and embedded in LRWhite. Ultrathin sections were contrasted with uranyl acetate and lean citrate, and examined by electron microscopy LEO 912 AB (Zeiss, Germany).

Immature seeds from wild-type and transformants at the two-WAFs were harvested, and fixed overnight in 1.25% glutaraldehyde and 2% paraformaldehyde in 50 mM PBS at 4°C as previously described (Kim et al., 2013 (link); Lee et al., 2015 (link)). After the fixed specimens were washed in PBS, dehydrated in a graded series (to 50% from 100%) of ethanol and embedded in Epon 812, the specimens were sliced to ultrathin sections with an ultramicrotome (Leica). Sections were stained with a solution of uranyl acetate and lead citrate and observed with Transmission Electron Microscopy (TEM; LEO912AB, Carl Zeiss, Jena, Germany). To observe the morphological changes of the mature seeds, germinated seeds and starch granules, the seeds were transversely and vertically sectioned. Photographs of the samples were taken using a light microscope and a scanning electron microscope (SEM).