title: Novel approaches for two and three dimensional multiplexed imaging of osteocytes.
abstract: Although osteocytes have historically been viewed as quiescent cells, it is now clear that they are highly active cells in bone and play key regulatory roles in diverse skeletal functions, including mechanotransduction, phosphate homeostasis and regulation of osteoblast and osteoclast activity. Three dimensional imaging of embedded osteocytes and their dendritic connections within intact bone specimens can be quite challenging and many of the currently available methods are actually imaging the lacunocanalicular network rather than the osteocytes themselves. With the explosion of interest in the field of osteocyte biology, there is an increased need for reliable ways to image these cells in live and fixed bone specimens. Here we report the development of reproducible methods for 2D and 3D imaging of osteocytes in situ using multiplexed imaging approaches in which the osteocyte cell membrane, nucleus, cytoskeleton and extracellular matrix can be imaged simultaneously in various combinations. We also present a new transgenic mouse line expressing a membrane targeted-GFP variant selectively in osteocytes as a novel tool for in situ imaging of osteocytes and their dendrites in fixed or living bone specimens. These methods have been multiplexed with a novel method for labeling of the lacunocanalicular network using fixable dextran, which enables aspects of the osteocyte cell structure and lacunocanalicular system to be simultaneously imaged. The application of these comprehensive approaches for imaging of osteocytes in situ should advance research into osteocyte biology and function in health and disease.
authors: Department of Oral and Craniofacial Sciences, School of Dentistry, University of Missouri, Kansas City, 615 E. 25th Street, Kansas City, MO 64108, USA; Biomedical Sciences Department, Oakland University William Beaumont School of Medicine, 414 O'Dowd Hall, Rochester MI, 48309, USA; Medical Physiology Department, Assiut University School of Medicine, 71516 Asyut, Egypt.
authors: Suzan A
authors: SA
authors: Kamel-ElSayed
authors: Department of Oral and Craniofacial Sciences, School of Dentistry, University of Missouri, Kansas City, 615 E. 25th Street, Kansas City, MO 64108, USA.
authors: LeAnn M
authors: LM
authors: Tiede-Lewis
authors: Department of Oral and Craniofacial Sciences, School of Dentistry, University of Missouri, Kansas City, 615 E. 25th Street, Kansas City, MO 64108, USA; Department of Biomedical Sciences, Texas A&M University Baylor College of Dentistry, 3302 Gaston Ave., Dallas, TX 75246, USA.
authors: Yongbo
authors: Y
authors: Lu
authors: Department of Oral and Craniofacial Sciences, School of Dentistry, University of Missouri, Kansas City, 615 E. 25th Street, Kansas City, MO 64108, USA.
authors: Patricia A
authors: PA
authors: Veno
authors: Department of Oral and Craniofacial Sciences, School of Dentistry, University of Missouri, Kansas City, 615 E. 25th Street, Kansas City, MO 64108, USA. Electronic address: dallass@umkc.edu.
authors: Sarah L
authors: SL
authors: Dallas
mesh_terms: D000818:Animals; D003952:Diagnostic Imaging / Q000379:methods*; D016326:Extracellular Matrix Proteins / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D010011:Osteocytes / Q000166:cytology*
chemical_list: C513308:Dmp1 protein, mouse; D016326:Extracellular Matrix Proteins; D049452:Green Fluorescent Proteins
keywords: 3D imaging; Bone histology; Collagen; Confocal imaging; Osteoblasts; Osteocytes