Intermediate intensity of fluorescent signal was detected from the bowel and skin (Fig. demonstrated that IGF-1R antibody was able to bind to IGF-1R on the cell membrane. Fluorescent IGF-1R antibody injected into the mouse tail vein made subcutaneous tumors and orthotopic tumors become fluorescent. The intensity of fluorescence from the tumor was stronger than that from surrounding normal tissues. Histochemical examination confirmed that the tumor was located inside the gallbladder and adjacent liver parenchyma of mice. Conclusions Our study Keratin 5 antibody showed that a fluorescent IGF-1R-targeted antibody could help detect gallbladder tumors. Tumor-specific imaging technology can be applied to endoscopy, laparoscopy, and robotic surgery for better management of gallbladder cancer. Keywords: Gallbladder neoplasms, Insulin-like growth factor-1 receptor, Imaging INTRODUCTION Gallbladder cancer is a potentially fatal cancer responsible for 1.7% of all cancer mortality.1 Because it is difficult to distinguish gallbladder cancer from benign gallbladder disease, many cases are diagnosed incidentally after cholecystectomy.2 Incidental gallbladder cancer represents between 1.6% and 50% of all gallbladder cancers. Incidental gallbladder cancer detection raises several medical problems. First, a second intervention is needed to ensure complete surgical Daidzein resection and tumor staging. In addition, unexpectedly resecting gallbladder cancer may cause cancer cell dissemination.3 This is one of the many reasons that gallbladder cancer is so deadly; thus, it is important to develop a better diagnostic method for gallbladder cancer to ensure timely treatment. Fluorescent imaging technology may help to diagnose gallbladder cancer. Previous studies reported that fluorescent imaging could improve tumor visualization and surgical Daidzein outcomes of colon cancer using fluorescent carcinoembryonic antigen antibody.4 Type I insulin-like growth factor-1 receptor (IGF-1R), a transmembrane tyrosine kinase expressed in pancreatic and colon cancers, can be a potential target for fluorescent imaging.5,6 Gallbladder cancer might be another cancer for which IGF-1R-targeted fluorescent imaging is useful. In this study, we sought to demonstrate that fluorophore-conjugated IGF-1R-targeted antibodies could be used to visualize gallbladder cancer in orthotopic tumor mouse models. MATERIALS AND METHODS 1. Gallbladder cancer cell lines The human bile duct cancer cell lines SNU-308, SNU-478, and SNU-1196 (Korean Cell Line Bank, Seoul, Korea) were maintained in RPMI 1640 and Minimum Essential Medium with Earles Balanced Salts medium supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), and antibioticCantimycotic solution (Gibco). All cells were cultured at 37C in a 5% CO2 incubator. 2. Mice Balb/c nude mice (Central Lab. Animal, Inc., Seoul, Korea), 4 to 5 weeks old, were used in the study. Mice were kept in a barrier facility under high efficiency particulate air filtration and fed with an autoclaved laboratory rodent diet. All mouse surgical procedures and imaging were performed after anesthetization by intramuscular injection of 60% tiletamine and zolazepam, and 40% xylazine HCl (0.02 mL). Animals received antibiotics immediately prior to surgery and once a day over the next 3 days. The maximum tumor size allowed was 2 cm. The condition of the animals was monitored every day. CO2 inhalation was used for euthanasia. This study was approved by and performed in accordance with the guidelines of the Yonsei University Health System Institutional Animal Care and Use Committee (IACUC number: 2020-0015). 3. Daidzein Antibody-dye conjugation Mouse monoclonal antibodies to IGF-1R (clone 24-31; Thermo Scientific, Rockford, IL, USA) were conjugated with DyLight 650 dyes (Thermo Scientific) after removing bovine serum albumin with PierceTM Antibody Clean-up Kit (Thermo Scientific) per manufacturer specifications.5,6 4. Western blotting Cell lysates were extracted in lysis buffer containing 70 mM -glycerophosphate, 0.6 mM sodium orthovanadate, 2 mM MgCl2, 1 mM ethylene glycol tetraacetic acid, 1 mM DTT (Invitrogen, Grand Island, NY, USA), 0.5% Triton-X100, 0.2 mM phenylmethylsulfonyl fluoride, and 1% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Lysates were separated via sodium dodecyl sulfateCpolyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% (w/v) non-fat dry milk and probed with.