title: Intensive Immunofluorescence Staining Methods for Low Expression Protein: Detection of Intestinal Stem Cell Marker LGR5.
abstract: Leucine-rich repeat-containing G-protein coupled receptor 5, or LGR5, is a molecule that recognizes stem cells in multiple organs and also in colon cancer. Previously, we have developed monoclonal antibodies specific to LGR5 protein that can be used for immunofluorescence staining, but because a very low level of LGR5 protein is expressed, the visualization technique needed to be enhanced. To develop procedures to detect LGR5 protein in various specimens by immunofluorescence staining, we evaluated the Alexa-labeled streptavidin biotin (LSAB), the Qdot, and the tyramide methods. The detection sensitivity was highest in the tyramide method followed by the Qdot method, whereas subcellular localization of the protein was most clear in the Qdot method, because the Qdot method gave a high S/N ratio that could show a low background. Thus, the tyramide method is superior to the Q-dot method for intensifying the signal of a low expression protein, and the Qdot method is superior to the tyramide method for identifying the subcellular localization of the target protein. The results of the present study will be helpful in providing more insight into the pathophysiological roles of LGR5-positive cancer stem cells and in developing therapeutic approaches for targeting cancer stem cells.
authors.affiliation: Research Division, Chugai Pharmaceutical Co., Ltd. , 1-135, Komakado, Gotemba, Shizuoka 412-8513, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Yamazaki
authors.affiliation: Research Division, Chugai Pharmaceutical Co., Ltd. , 1-135, Komakado, Gotemba, Shizuoka 412-8513, Japan.
authors.forename: Atsuhiko
authors.initials: A
authors.lastname: Kato
authors.affiliation: Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University , 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Zaitsu
authors.affiliation: Chugai Research Institute for Medical Science, Inc , 1-135, Komakado, Gotemba, Shizuoka 412-8513, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Watanabe
authors.affiliation: Department of Molecular Oncology, Graduate School of Medical Sciences, Kyushu University , 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Iimori
authors.affiliation: Forerunner Pharma Research Co., Ltd. , 4-2-16, Komaba, Meguro-ku, Tokyo 153-8904, Japan.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Funahashi
authors.affiliation: Department of Molecular Oncology, Graduate School of Medical Sciences, Kyushu University , 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kitao
authors.affiliation: Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University , 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Saeki
authors.affiliation: Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University , 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Eiji
authors.initials: E
authors.lastname: Oki
authors.affiliation: Research Division, Chugai Pharmaceutical Co., Ltd. , 1-135, Komakado, Gotemba, Shizuoka 412-8513, Japan.
authors.forename: Masami
authors.initials: M
authors.lastname: Suzuki
keywords: Alexa; LGR5; Qdot; immunofluorescence; tyramide