Bone Sectioning Technique for 3D Confocal Image Resolution and Capture of Dye-Loaded Nanotherapeutics

ABSTRACT Purpose:Capturing detailed images of bone architecture has unique challenges and conventional procedures have proved to be insufficient for molecular 3D imaging. Furthermore, traditional 2D immunohistochemistry provides limited information for assessing therapeutic localization in the bone. In addition, techniques such as thin paraffin sections visualized by immunofluorescence microscopy or transmission electron microscopy, require prolonged exposure to damaging decalcification reagents. These chemicals have destructive effects on bone morphology and limit the capture of proteins. The objective of this project was to develop an adapted protocol for bone tissue preparation prior to sectioning and immunohistochemical (IHC) staining. This method enables ultra-thick sections for enhanced Z-stacking, enables the generation of high-resolution 3D images that map the bone tissue, and provides oseo-spatial detection of our dye-loaded nanotherapeutics. Methods:Bones were decalcified then incubated in cryoprotectant before emersion in the embedding solution. Samples were frozen at -80. Ultra-thick sections were made on a Thermo Fisher Cryostar NX70 Cryostat (75 – 100 m) and placed on polar slides. Immunohistochemical staining was applied to the slides, which were imaged with a Zeiss LSM 510 confocal microscope. Our therapeutic was labeled with near fluorescent dye.Results:High-fidelity, 3D images of mouse tibia and femur were imaged. Furthermore, visualization of nuclear staining, bone epithelial cells, and the fluorescently labeled therapeutics were easily detected. Thick sectioning provided us with a more robust, tomographic image, allowing for more thorough mapping and analysis of the nanotherapeutics in the bone. Conclusions:Our modified protocol for processing and imaging bone is an effective approach to bone handling, confocal imaging, and detecting bone and dye-labeled nanotherapeutics. This approach will provide benefits for facilitating our understanding of the significance that drug localization has on the bone microenvironment and its impact on therapeutic efficacy.