Phosphate buffer

Immunofluorescence staining was performed to confirm the pluripotency of AB-MSC spheroids. This protocol was consistent with that reported previously [14 (link)]. In brief, spheroids were collected and solidified in iPGell (Genostaff, Tokyo, Japan) according to the manufacturer’s protocol. The samples were fixed with 4% paraformaldehyde in phosphate buffer (Wako), embedded in paraffin, and sectioned at a thickness of 8 μm. Primary antibodies for pluripotency markers included Oct4 (ab19857), Sox2 (ab79351), KLF4 (ab216875), and cMyc (ab32072). The primary antibodies for neuronal markers were neurofilament medium (NF-M, ab7794), βIII tubulin (ab78078), NeuN (ab12763), and GFAP (ab279290). The cell proliferation marker Ki67 (ab15580) and apoptosis marker Caspase3 (ab13585) were also analyzed. The secondary antibodies were goat anti-rabbit IgG (Alexa Fluor 647, ab150079) and goat anti-mouse IgG (Alexa Fluor 488, ab150113). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, ab104139). All antibodies were purchased from Abcam (Cambridge, UK). Positive fluorescent staining was observed using an inverted microscope (Olympus IXplore Spin microscope; Tokyo, Japan).

A total of 147 oral Kampo prescriptions, which are covered by health insurance in Japan, were purchased from Ohsugi Pharmaceutical Co., Ltd. (Osaka, Japan), Kracie Holdings, Ltd. (Tokyo, Japan), Kotaro Pharmaceutical Co., Ltd. (Osaka, Japan), Sanwa Shoyaku Co., Ltd. (Tochigi, Japan), Taikoseido Pharmaceutical Co., Ltd. (Hyogo, Japan), Tsumura & Co. (Tokyo, Japan), and Toyo-Kampo Pharmaceutical Co., Ltd. (Osaka, Japan), respectively. The Albumin Glycation Assay Kit, Glyceraldehyde (Cat. No. AAS-AGE-K01) was purchased from Cosmo Bio Co., Ltd. (Tokyo, Japan). Sodium azide was purchased from Sigma-Aldrich Japan Co. (Tokyo, Japan). Ephedrae herba was purchased from Tanuma Shokai Co., Ltd. (Chiba, Japan) (Lot. 131017). Methylglyoxal and Glyceraldehyde were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). An amount of 0.1 moL/L Phosphate buffer, acetic acid, and dimethyl sulfoxide were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). LC-MS-grade acetonitrile, methanol, formic acid, and distilled water were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan).

Immunofluorescence was conducted according to the protocol in our previous publication.^{10 (link)} In brief, spheroids were collected and solidified by iPGell (Genostaff Co., Ltd., Tokyo, Japan) according to the manufacture's instruction. The samples were fixed with 4% paraformaldehyde in phosphate buffer (pH 7.4; FUJIFILM Wako Pure Chemical Corporation), dehydrated, and embedded in paraffin. Sections (8 μm in thickness) were cut.
After deparaffinization and rehydration, the sections were incubated with primary antibodies against pluripotency markers: Nanog (1:100; No. ab80892; Abcam), Sox2 (1:250; No. ab97959; Abcam), octamer-binding transcription factor 4 (Oct4; 1:250; No. ab19857; Abcam), and SSEA1 (1:100; No. ab16285; Abcam) overnight at 4°C. The sections were then washed in PBS three times. Secondary antibodies conjugated to Alexa Fluor (ab150121 and ab150079; Abcam) were added for 2 h at room temperature in the dark. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (ab104139; Abcam) for 30 min. The images were captured with a fluorescence microscope (KEYENCE BZ-X710; Keyence, Osaka, Japan).

Cells were grown in 35 mm dishes. After 10–12 h, cells were pre-fixed in 2.5% glutaraldehyde solution for electron microscopy (Wako, Japan), 2% PFA, and 0.1 M phosphate buffer (Wako, Japan) for 2 h at room temperature, washed three times with PBS, and dehydrated in an ascending gradual series of ethanol (50%, 70%, 90%, 95%, and 100%) for 8 min each. Cells were then mounted in an epoxy resin using propylene oxide to aid infiltration, which was hardened at 60 °C for 24 h. Ultrathin sections were taken using a diamond knife, mounted on copper mesh grids, stained with uranyl acetate, and imaged using a Topcon EB-002B transmission electron microscope.

Benzaldehyde-2-sulfonic acid sodium salt (BS), chloranil, 2-methylresorcinol (2,6-dihydroxytoluene) (DHT), and 2,4,6-trihydroxybenzaldehyde (THBA) were purchased from Tokyo Chemical Industry (Tokyo, Japan). Pyrrole (Pyr), pyrogallol (1,2,3-trihydroxybenzene, THB), acetyl chloride, triethylamine, p-toluenesulfonic acid monohydrate (p-TS), tetrabutylammonium hexafluorophosphate, acetonitrile, and phosphate buffer were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). The Pyr monomer was purified via distillation prior to use.

NYMV strain tick 39 was rescued by a reverse genetics system as described previously [14 (link)] with slight modifications. First, 293T cells were seeded into a 6-well plate and later transfected with pNYMV (4 μg), pCA-NYMV-N (0.5 μg), pCA-NYMV-P (0.2 μg), and pCA-NYMV-L (0.2 μg). The plasmids were kindly provided by Peter Stäheli. After observing the cytopathic effect, the supernatant was transferred to Vero cells. Then, after 10 days, the supernatant was collected and centrifuged at 2500× g for 10 min, and the supernatant was filtered using a 0.22 μm syringe filter, aliquoted to tubes, and stored at −80 °C until use. The virus stock was titrated using Vero cells.
U-2 OS cells (1 × 10⁵) were seeded into 12-well plates with 15 mm coverslips (MATSUNAMI GLASS) and were infected with 7.5 × 10³ Tissue Culture Infectious Dose 50 (TCID₅₀) of NYMV. After 24 h, the cells were fixed with 4% paraformaldehyde in phosphate buffer (Wako, Osaka, Japan) for 10 min and subjected to immunofluorescence assay (IFA).

Cellular samples were fixed in 0.9% NaCl, 1% glutaraldehyde (Wako) for 1 h at 4 °C. Cells were pelleted and re-suspended in 1 mL of a 37 °C pre-warmed 1.1% agarose solution (Bio-Rad). After centrifugation (3000 rpm at 37 °C), pellets were placed at 4 °C and cut into small specimens, washed in 5% sucrose PBS (Wako) and post-fixed with 5% sucrose, 1.5% osmic acid (Nisshin EM) in 100 mM phosphate buffer (Wako) pH 7.3, for 1 h at 4 °C. Post-fixed specimens were washed in 5% sucrose PBS and dehydrated in a graded series of ethanol (Wako) and propylene oxide (Wako), then embedded in epoxy resin EPON 812 (TAAB Laboratories). Ultrathin sections were obtained with an ultra-microtome (Leica EM UC7) and subjected to double staining with a uranyl-acetate (Wako) and lead-citrate (Nacalai Tesque). Samples were observed under a JEM-1400Plus Electronic Microscope (JEOL). TEM were analyzed by collecting images of at least 3 representative areas (RA) at magnification of ×500. One RA contained 10–20 nucleated cells. Then representative images were taken at higher magnifications.