D sucrose

2-Hydroxyethyl methacrylate (2-HEMA) was supplied by Merck (Darmstadt, Germany); ethylene glycol dimethacrylate (EGDMA), Dulbecco’s Modified Eagle’s Medium (DMEM) and dichlorodimethylsilane were from Sigma-Aldrich (Steinheim, Germany); N-(3-aminopropyl) methacrylamide hydrochloride (APMA) was from Polysciences Inc. (Warrington, PA, USA); D(+)-sucrose (99.7%) and 2,2′-azo-bis(isobutyronitrile) (AIBN) from Acros (Geel, Belgium). The Cy3 Ab Labeling Kit was supplied from Amersham/GE Healthcare (Munich, Germany). The AAV Titration ELISA was from Progen (Heidelberg, Germany) and the IGF-1 ELISA kit (Insuline like Growth Factor 1) from R&D Systems (Nordenstadt, Germany); the Beta-Glo^® Assay System was from Promega (Mannheim, Germany); the β-gal staining kit and cell proliferation reagent WST-1 were obtained from Roche Applied Science (Mannheim, Germany). Vectastain ABC HRP kit (Peroxidase, Standard) and Biotynilated Dolichos Biflorus Agglutinin (DBA) were from Vector Laboratories (Burlingame, CA, USA). The antibody anti-IGF-I (AF-291-NA) was from R&D Systems (Nordenstadt, Germany). Ultra-pure water (resistivity > 18.2 MΩ·cm) was obtained by reverse osmosis (MilliQ^®, Millipore Ibérica, Madrid, Spain) and water for cell culture was from Sigma-Aldrich (Steinheim, Germany). All other reagents were analytical grade.

Polyvinyl alcohol (78% hydrolyzed, M_w = 6,000 Da) and D (+)-Sucrose (≥99% Ultra-Pure Grade, M_w = 342.29 Da) were purchased from Acros Organics (New Jersey, United States). FITC-Insulin was provided by ZhongKeChenYu Biotech Co. Ltd (Beijing, China). Streptozotocin was provided by AbMole Bioscience (Shanghai, China). Fluorescence microscopy (Olympus SZX7) was obtained from Micro-Shot Technology Co., Ltd (Guangzhou, China). Model ESM301 Force Test Stand and Series 5 Digital Force Gauge were purchased from Mark-10 Corporation (Copiague, United States). In Vivo Imaging Systems (IVIS Spectrum) was purchased from Caliper Life Sciences (Massachusetts, United States). All chemicals were used as received without additional treatment.

An alcohol solution (20% v/v) was prepared by mixing 200-proof absolute alcohol (Cat#B28184; Fisher Scientific, Pittsburgh, PA, USA) with water. Additionally, a sucrose solution (10% w/v) was prepared by dissolving sucrose (D-sucrose; Fisher Scientific) in water. For real-time micro-endoscopic in vivo imaging of cholinergic interneurons (CIN), adeno-associated virus (AAV; serotype 1) was employed, utilizing the FLEX switch system coupled with the calcium indicator GCaMP6s under the regulation of the human synapsin 1 promoter (GCaMP; AAV1.Syn.Flex.GCaMP6s.WPRE.SV40; 2 × 1013 GC/mL, diluted to 50%; Addgene, Cambridge, MA, USA). GCaMP, which stands for genetically encoded calcium indicator, is composed of several components, including the calcium-binding protein calmodulin (CaM), a peptide sequence derived from myosin light-chain kinase known as M13, and green fluorescent protein (GFP). When there are fluctuations in intracellular free calcium caused by neuronal activity, it triggers changes in the structure of the CaM–M13 complex, resulting in increased fluorescence emitted by GFP. This mechanism allows for the observation of neuronal activity in vivo using microscopy. All viral preparations were promptly aliquoted upon receipt and stored at −80 °C in a deep freezer. Moreover, all solutions were freshly prepared immediately before usage.

Anti-FLAG mouse monoclonal antibody was purchased from Stratagene (Cat # 200472). Anti-HA mouse monoclonal antibody was purchased from Thermo Fisher Scientific (Cat #26183). Anti-GAPDH, HRP-conjugated antibody was purchased from Santa Cruz Biotechnologies (Cat #sc-365062 HRP). Goat anti-mouse and anti-rabbit HRP conjugated secondary antibodies were purchased from Azure Biosystems (Cat #AC2115) or Cell Signaling Technologies (Cat #7074S), respectively. Antibodies raised against VACV proteins A17 and A10 were gifts from Dr. Bernard Moss [65 (link)–67 (link)]. Antibodies raised against VACV protein E3 were a gift from Dr. Yan Xiang [68 (link)]. Cycloheximide(Cat#01810) was purchased from Sigma. Phenol:Chloroform:Isoamyl Alcohol (Cat#15593031) was purchased from Invitrogen. qPCR mix (Cat#QP005) was purchased from GeneCopoeia. SuperScript III Reverse Transcriptase (Cat#18-080-044), Trizol reagent (Cat#15-596-018), RNase inhibitor (Cat#AM2696), DTT (Cat#BP172) and D-Sucrose (Cat#BP220-212) were purchased from Fisher Scientific.