Sample preparation for high-resolution 3D confocal imaging of mouse skeletal tissue

Abstract

High-resolution confocal imaging is a vital tool for analyzing the 3D architecture and detailed spatial distribution of cells in situ. However, imaging of skeletal tissue has remained technically challenging because of its calcified nature. Here we describe a protocol that allows high-resolution imaging of skeletal tissue with preservation of cellular morphology and tissue architecture. The procedure involves tissue fixation, decalcification and cryosectioning of the mouse skeletal tissue to generate thick sections. The thick sections generated by this procedure are not only compatible with the analysis of genetically expressed fluorescent proteins but they also preserve antigenicity, thus enabling diverse combinations of antibody labeling. Further, this procedure also permits other fluorescence techniques such as TUNEL and ethynyl deoxyuridine (EdU) incorporation assays. Images resulting from the confocal imaging can be assessed qualitatively and quantitatively to analyze various parameters such as distribution and interrelationships of cell types. The technique is straightforward and robust, highly reproducible and can be completed in ∼11 d.

MiR-497∼195 cluster regulates angiogenesis during coupling with osteogenesis by maintaining endothelial Notch and HIF-1α activity

“…The bones were then incubated in 20% sucrose and 2% polyvinylpyrrolidone (PVP) solution for 24 h, as described previously333435. Finally, we embedded the tissues in 8% gelatin (porcine; Sigma, G2500) in presence of 20% sucrose and 2% PVP (Sigma, PVP360).…”

Blood vessel formation and function in bone

“…Such early studies also suggested that the organization of the bone vasculature is similar in different mammalian species, including rats, rabbits, guinea pigs and humans (Trueta and Morgan, 1960). In recent years, various technological advancements, such as the identification of cell type-specific markers, transgenic florescent reporters, imaging by confocal and two-photon microscopy, microcomputed tomography (micro-CT), three-dimensional (3D) reconstruction of imaging data, and improved protocols for tissue processing and immunostaining, have advanced our understanding of the organization of the bone vasculature and its functional specialization (Acar et al, 2015;Kunisaki et al, 2013;Kusumbe et al, 2014Kusumbe et al, , 2015Roche et al, 2012).…”

Confocal/two-photon microscopy in studying colonisation of cancer cells in bone using xenograft mouse models

“…The comprehensive methodology from collecting fresh bone tissues to cryosectioning and immunostaining has been described previously. 28 Imaging bone samples with confocal/two-photon microscopy Basic microscope settings. The bone structure can be visualised by SHG using a Chameleon laser at 900 nm (Coherent, Santa Clara, CA, USA), while Vybrant-DiD labelled cancer cells can be visualised using a 633 nm HeNe laser and Vybrant DiI/CM-DiI with a 543 nm HeNe laser.…”