E 1010

Samples were cut into pieces (5 mm thickness) and fixed with 2.5% glutaraldehyde in 0.2 M phosphate buffer for 2 h at room temperature. The specimens were washed in deionized distilled water three times for 15 min each time. Then, the specimens were washed in 50% ethanol (v/v) two times for 5 min, in 70% ethanol for 10 min, in 80% ethanol for 10 min, in 90% ethanol for 10 min, and in100% ethanol two times for 20 min. All specimens were coated with gold using an ion sputter (E-1010, Hitachi High Technologies Co., Tokyo, Japan) and observed using a low-vacuum scanning electron microscope (LV-SEM) operating at 15 kV.

Mice were perfused with 2% PFA and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). Tissues were dissected and further fixed for 2 hours at RT and treated with 1% OsO4 in 0.1 M cacodylate buffer followed by 0.5% uranyl acetate in water. The samples were dehydrated and embedded in Epon, and thin sections were post-stained with uranyl acetate and lead citrate as described previously (Harada et al., 1990 (link)). Sections were then studied under an electron microscope (model 1010; JEOL, Tokyo, Japan) at 80 kV.
For scanning EM, fresh tissues were dissected out and fixed for 4 hours in 2% PFA and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). Tissues were further fixed in 1% tannic acid in 0.1 M cacodylate buffer for 2 hours and 1% OsO₄ in 0.1 M cacodylate buffer for 1 hours. Tissues were then transferred to 50% DMSO in water, frozen and fractured in liquid nitrogen. The samples were substituted in t-butyl alcohol, freeze-dried and sputter-coated with Pt-Pd (E-1010; Hitachi High-Technologies Corporation, Tokyo, Japan). Samples were viewed at 15 kV using scanning electron microscopy (S-4100; Hitachi High-Technologies Corporation).

The microstructure of HSBC aerogels was observed using a field-emission scanning electron microscope (FE-SEM: S-4500, Hitachi High-Technologies Corporation, Minato-ku, Tokyo, Japan) with an acceleration voltage of 10 kV. For the pretreatment prior to FE-SEM observation, deposition of Pt–Pd was performed by ion sputtering (E-1010, Hitachi High-Technologies Corporation).
To analyze the composition and the crystal structure of the obtained HSBC aerogels, attenuated total reflection Fourier transform infrared (ATR-FTIR) measurement was carried out. The ATR-FTIR spectra were measured using a FTIR spectrophotometer (Spectrum One, Perkin Elmer, Billerica, MA, USA) equipped with a universal ATR sampling accessory. All measurements were carried out with a nominal spectral resolution of 8 cm⁻¹ in transmittance mode and 24 scans.
The weight of encapsulated activated carbon was measured using a microgram balance (Sartorius, MSE3.6P000DM, readability: 1 μg). As a pre-treatment, all samples were dried sufficiently at 50 °C for 1 day. The weight of the activated carbon was determined by the following equation:
The HSBC gel was prepared on the same day, using the same cell suspension as the preparation of the activated-carbon-encapsulated HSBC gel.

The fixed samples were dried at room temperature for 24 h, and then gold was vapor-deposited on the sample using an ion sputterer (E-1010, Hitachi High-Technologies Corp., Tokyo, Japan). The surface of the inner skin of ‘Porotan’ chestnuts was observed using a scanning electron microscope S-3000N (Hitachi Science Systems, Ltd., Tokyo, Japan). The microscope was operated at an acceleration voltage of 20 kV.

Images of raw fibers, BC samples, and fibers in the electrospun webs were acquired using a scanning electron microscope (TM3000, Hitachi High-Tech Co., Tokyo, Japan) at an accelerating voltage of 15 kV. Prior to analysis, the samples were fixed on the sample holder using conductive C tape and coated with a layer of Au sprayed by an ion sputter coater (E-1010, Hitachi High-Tech Co., Tokyo, Japan) as a pretreatment. Images of the webs were obtained under various LES conditions. For each image, diameters of the fibers were measured at 200 different positions using a software application (ImageJ version 1.8.0, U. S. National Institutes of Health, Bethesda, MD, USA) to evaluate the average diameter and CV.