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18 protocols using sigma vp scanning electron microscope

1

Micromorphology and Anatomy of Plant Nectaries

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Shoot apices and young leaves were fixed in formalin–acetic acid–alcohol (FAA) solution for 24 h [62 ] and buffered neutral formalin in 0.1 M sodium phosphate buffer (pH 7.0) for 48 h [63 ]. For the micromorphological study, mature nectaries were isolated, dehydrated in a graded ethanol series, dried by the critical point method, mounted on aluminium stub, and sputter-coated with gold, with subsequent observation in a Zeiss Sigma VP scanning electron microscope (Carl Zeiss, Oberkochen, Germany).
For anatomical analyses, shoot apices and young leaves were isolated, dehydrated through a tertiary butyl alcohol series [62 ], embedded in Paraplast® (Leica Microsystems Inc., Heidelberg, Germany), and serial-sectioned at a 12 µm thickness on a Leica RM2145 rotary microtome. Longitudinal and transverse sections were stained with astra blue and safranin O [64 ] and the slides were mounted with resin Permount (Fisher Scientific, Pittsburgh, PA, USA). Observations and photographs were performed using a Leica DMLB light microscope.
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2

Characterization of CNF and TOCNF Hydrogels

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CNF and TOCNF hydrogels were diluted to final concentrations of 5 mg/l and 8 mg/l, respectively. 25 μl of each dispersion was cast on a mica support and dried for an hour at 50 °C. The dry samples were imaged with AFM (Nanoscope IIIa Multimode scanning probe microscope from Digital Instruments Inc., USA). Images were obtained by using tapping mode in air with silicon cantilevers. For filament imaging, SEM was used with magnifications 10300x and 35490x (Zeiss Sigma VP scanning electron microscope, Carl Zeiss Microscopy Ltd, Cambridge, UK). The operating voltage was either 3 kV or 2 kV and the working distance either 2.5 mm or 2.6 mm. Prior to imaging, the sample was sputtered with carbon followed by gold-palladium coating. Optical microscope image between crossed polarisers was obtained with a polarising microscope Leica DM4500 P equipped with a Leica DFC420 camera (Leica Microsystems, Germany).
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3

High-Resolution Volumetric Imaging of Neuronal Ultrastructure

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Samples were imaged in 2 instruments. High-resolution montaged sets were generated in a Thermo Fisher Scientific (FEI) Volumescope2 system. Images were acquired at 2.0 kV in high-vacuum mode using T1 detector and Volumescope stage. Fields were imaged at 5 nm/pixel magnification, and images of 3,000 × 5,000 pixels wide were stitched to produce slices of 170 μm. Sets of 300–600 slices were collected at cut thicknesses of 50–60 nm, using MAPS2 software (Thermo Fisher Scientific). Montaging sets of small-image tiles reduced sample/beam drift. Samples were also imaged with a Zeiss Sigma VP scanning electron microscope (Carl Zeiss Microscopy) with a 3View in chamber ultramicrotome (Gatan/Amaritech). These were imaged at 2.0 kV, using a 30 μm aperture in high-vacuum mode. Large fields of 10,000 × 12,000 pixels covered an area of 130 μm wide and did not require stitching. A series of 300–500 slices was generated with 60 nm slices and 7 nm/pixel resolution. The high-resolution sets were used to follow fine structures, while lower-resolution sets provided more rapid analysis of synaptic bouton occupancy of neuronal somas.
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4

Micromorphology and Anatomy of Plant Nectaries

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Shoot apices and young leaves were fixed in formalin–acetic acid–alcohol (FAA) solution for 24 h [62 ] and buffered neutral formalin in 0.1 M sodium phosphate buffer (pH 7.0) for 48 h [63 ]. For the micromorphological study, mature nectaries were isolated, dehydrated in a graded ethanol series, dried by the critical point method, mounted on aluminium stub, and sputter-coated with gold, with subsequent observation in a Zeiss Sigma VP scanning electron microscope (Carl Zeiss, Oberkochen, Germany).
For anatomical analyses, shoot apices and young leaves were isolated, dehydrated through a tertiary butyl alcohol series [62 ], embedded in Paraplast® (Leica Microsystems Inc., Heidelberg, Germany), and serial-sectioned at a 12 µm thickness on a Leica RM2145 rotary microtome. Longitudinal and transverse sections were stained with astra blue and safranin O [64 ] and the slides were mounted with resin Permount (Fisher Scientific, Pittsburgh, PA, USA). Observations and photographs were performed using a Leica DMLB light microscope.
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5

Ultrastructural Analysis of Axonal Swelling

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Enucleated eyes were immersed in 0.1 M PBS containing 4% PFA (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) and 0.5% glutaraldehyde (Fujifilm Wako Pure Chemical Corporation) for 12 hours at 4°C. ON sections were cut on a vibratome (Leica), and the resulting specimens were stained with 0.4% OsO4, uranyl acetate, and lead aspartate, followed by embedding in epon resin (Electronic Microscopy Sciences, Hatfield, PA, USA). SBF-SEM images were obtained with a Sigma VP scanning electron microscope (Carl Zeiss, Oberkochen, Germany). Axonal swelling was defined as axonal segments with a diameter greater than twice that of adjacent segments, with prominent accumulation of membranous organelles and a lack of continuous cytoskeletal bundle profiles.
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6

Correlating SEM with DAPI-Stained Cells

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Fixed cells in metaphase were dropped onto Zeiss coverslips with fiducial markings. Images of DAPI-stained cells were captured with a Zeiss 880 Airyscan confocal microscope, and the locations of select cells were stored using the Shuttle and Find feature of the ZEN Black software. To correlate SEM with the DAPI-stained acquired images, the coverslip was briefly washed with ddH2O and stained with 2% uranyl acetate for 2 minutes. The coverslip and holder were then loaded into the SEM and the same previously imaged DAPI-stained cells in metaphase were located using Shuttle and Find with ZEN Blue software. Images were captured using a Zeiss Sigma VP Scanning Electron Microscope and correlated with light microscope images.
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7

Wax Suspension SEM Imaging

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The
wax suspension was
dropped on silica and dried overnight. A Au/Pd coating of 4 nm was
sputtered on the sample before observation with a Zeiss Sigma VP scanning
electron microscope (SEM) operating at 2 kV. Several images were taken,
and the most representative one is shown.
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8

Aerogel Sample Preparation for SEM Imaging

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The specimen for SEM imaging were prepared using different sample preparation methods to obtain aerogels. GrowDex and agarose samples were plunge-freezed in liquid propane and subsequently lyophilized. The alginate sample was prepared using a supercritical carbon dioxide drying method. Matrigel, ovomucin, collagen and alginate-RGD samples were prepared using glutaraldehyde (3.5%) fixation for overnight. After washing the excess fixative with water, the specimen was dehydrated with a series of water/ethanol (70/30, 50/50, 30/70, 10/90 and 0/100 v/v) mixture by incubating 10 min in each solution. Finally, samples were chemically dried with hexamethyldisilazane (HMDS) by incubating in each solution for 10 min in 1:1 (v/v) ethanol: HMDS and 2 x HMDS.
SEM images were acquired with a Zeiss Sigma VP scanning electron microscope with an acceleration voltage of 1.0 to 1.5 kV. The aerogel sample was placed on the carbon tape and sputter-coated with Au/Pd with Emitech K950X/K350 or a Leica EM ACE600 high vacuum sputter coater. Sample preparation methods, coatings, and acceleration voltages for each matrix appear in Supplementary Table 1a.
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9

Spheroid Analysis: Imaging and Kinetics

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Analysis of cellular association and the growth kinetics of spheroids were accomplished by capturing brightfield images at regular time intervals and cell concentrations (575, 1425, 2285, 2850, 3700 cells/spheroid; 35 spheroids/well). Brightfield images were captured using a Cytation 3 Imaging Reader (BioTek, Winooski, VT). The 3D structures of the spheroids were observed using a Zeiss SIGMA VP Scanning Electron Microscope (Germany).
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10

Ultrastructural Analysis of Dendritic Spines

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For serial block-face scanning electron microscopy (SBF-SEM), P30 WT, Plxna2−/− and Sema5A−/− mice were perfused transcardially for 2 min with ice-cold PBS and then for 10 min with freshly prepared, ice-cold 4% PFA and 2.5% glutaraldehyde in 0.1 M Na+-cacodylate buffer (pH 7.4). Brains were dissected, postfixed in perfusion solution, and sectioned coronally in perfusion solution at 400 µm using a Leica vibrotome (TV1200). Brain slices that included the rostral hippocampus were fixed overnight in perfusion solution at 4°C and then processed for SBF-SEM according to instructions by Renovo Neural Inc. (Cleveland, OH). Briefly, tissue sections were rinsed in 0.1 M Na cacodylate buffer, stained with 0.1% tannic acid for 30 min that was followed by the staining regime developed by Renovo. Tissue was then dehydrated in ethanol and infiltrated and embedded in EPON 812. Approximately, 300 serial electron micrographs at 75-nm thickness were cut and imaged at a resolution of 7 nm/pix at Renovo Neural Inc., using a sigma VP scanning electron microscope (Carl Zeiss Microscopy, Jena, Germany) and a 3view door (Gatan Inc., Pleasanton, CA). For 3D reconstruction and quantification of dendritic segments, dendritic spine density, spine volume, spine head volume, and PSD surface, these structures were manually traced in individual sections using the PSD Reconstruct software (SynapseWeb).
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