S 520

Spermatozoa collected from the epididymides or oviduct were concentrated by centrifugation and resuspended in 2.5% glutaraldehyde and fixed for 3 h. After three washes in 0.1 M cacodylate buffer the samples were post-fixed in 1% osmium tetroxide for 1 h. After dehydration through a graded ethanol series, a drop of spermatozoa suspended in ethanol was placed on a glass cover slip, and the spermatozoa were allowed to settle. The samples were taken through critical point drying in CO₂ and coated with gold60 (link). Images were taken with a scanning electron microscope (SEM; Hitachi S-520, Japan). The morphometric parameters were measured from at least 30 spermatozoa.

The morphologies of the block copolymer composite ES nanofibers were examined using both SEM (Scanning Electron Microscope—Hitachi S-520, Tokyo, Japan) and an Optical microscope (MA-tek, Taipei, Taiwan). Before image characterization and analysis, SEM and EDS samples were coated with platinum and observed at an acceleration of 15 kV. EDS was employed for the elemental analysis of silver nanoparticle composites. The Optical microscope (OM) was primarily utilized to observe the various media layer fibers produced during different Electrospinning times. An Instron tensile tester (QC–H21B1-S00) was employed to characterize the stretchable conductors’ mechanical properties. The thermal stability of the composite ES nanofibers was analyzed using Thermogravimetric analysis (TGA, NETZSCH, Dubai, UAE). A custom-built automated two-probe measurement system connected to a Keithley 2400 Source Meter was used to measure the electrical conductivity of the stretchable conductor samples. At least ten samples were measured to calculate the average and enhance accuracy.

Rat gastric antrum, 0.5 cm × 0.5 cm in size, was rinsed in saline and then rapidly removed to 2.5% glutaraldehyde (PBS preparation) for fixation. Tissue samples were placed in 1% osmic acid, and then underwent gradual dehydration with alcohol, isoamyl acetate replacement, critical-point drying, and metal spraying. A scanning electron microscope (S-520, Hitachi Ltd., Tokyo, Japan) was used to observe gastric mucosa ultrastructure. Observed the gastric mucosa ultrastructure changes by pathologists, on the principle of a double blindness.

To observe the ultrastructure of the hydrogel scaffolds, the samples were coated with gold-palladium and examined under a scanning electron microscope (SEM; Hitachi S-520, Hitachi, Japan). The interior microstructures of the scaffolds were investigated using ImageJ (US National Institutes of Health, Bethesda, MD, USA). The distribution of the pore sizes, as well as the porosity, was assessed. The channels formed by PCL reverse mold were not included in the pore sizes and porosity. To examine the cell adhesion, the cell-scaffold constructs were prepared for SEM examination after 7 days of in vitro culture. The sample was fixed with 2.5% glutaraldehyde at 4 °C for 12 h then dehydrated using a graded ethanol series. A cross-section of the dried sample was coated with gold-palladium and examined under SEM.

The ECM scaffolds were cut into slices with a sharp blade. The interior cross sections and vertical sections were consecutively fixed using glutaric acid and osmic acid for 2 h and were dehydrated using a gradient of alcohol. Then, the samples were coated with gold-palladium and examined under scanning electron microscope to investigate the microstructures of the sections (SEM; Hitachi S-520, Japan).
The porosity was measured using the ethanol intrusion method10 (link). The volume of the absolute alcohol in a test tube was recorded as V_1. The scaffold was immersed in the test tube, and the volume of the absolute alcohol was recorded as V₂. After scaffold was taken out of the absolute alcohol, remaining alcohol was recorded as V₃. The porosity of the scaffold was, thus, E = (V₁ - V₃)/(V₂ - V₃). The scaffold was measured five times and the mean value is reported.
To measure the hydrating expansivity of the scaffold, a scaffold was completely immersed in deionized water for 10 min, and the weight was recorded as W. After the scaffold was taken out of deionized and dried in a vacuum drying oven at 50 °C, the scaffold weight was recorded as W₀. The scaffold was measured five times and the mean value is reported.

Heart tissue was fixed in 2% EM grade gluteraldehyde in 0.1M sodium cacodylate buffer pH 7.3 for 1 hour at room temperature, then stored in 0.1M sodium cacodylate buffer, pH 7.3, 0.2M sucrose until further processing. Fixed samples were chemically dehydrated in hexamethylsilazine, mounted on stubs and sputter-coated (Hummer VI Au/Pd 40/60) and examined with a high-resolution scanning electron microscope (Hitachi S-520) at an accelerating voltage of 20 kV equipped with a passive image capture system (Hitachi, Quartz PCI Version 6).

Spermatozoa collected from the oviduct were concentrated by centrifugation and resuspended in 2.5% (v/v) glutaraldehyde for 3 h. After washing in cacodylate buffer, the samples were postfixed in 1% (w/v) osmium tetroxide for 1 h. The samples were dehydrated through a graded ethanol series. A drop of spermatozoa suspended in ethanol was placed on a glass coverslip, and the spermatozoa were allowed to settle. The samples were taken through critical point drying in CO₂ and coated with gold (Sanders et al. 1975 (link)). Images were taken with scanning electron microscope (S-520; Hitachi, Tokyo, Japan).