Sigma vp scanning electron microscope

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.

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.

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.

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).