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Free keywords:
Mineralised tissue, Bone ultrastructure, Bone extracellular matrix, Controlled-angle microtome sectioning, Immunohistochemistry (IHC), Second harmonic generation (SHG), Confocal laser scanning microscopy (CLSM), Lacunar-canalicular network (LCN)
Abstract:
Histological processing of mineralised tissue (e.g. bone) allows examining the anatomy of cells and tissues as well as the material properties of the tissue. However, resin-embedding offers limited control over the specimen position for cutting. Moreover, specific anatomical planes (coronal, sagittal) or defined landmarks are often missed with standard microtome sectioning. Here we describe a method to precisely locate a specific anatomical 2D plane or any anatomical feature of interest (e.g. bone lesions, newly formed bone, etc) using 3D micro computed tomography (microCT), and to expose it using controlled-angle microtome cutting. The resulting sections and corresponding specimen's block surface offer correlative information of the same anatomical location, which can then be imaged using multi-scale microscopy. Moreover, this method can be combined with immunohistochemistry (IHC) to further identify any component of the bone microenvironment (cells, extracellular matrix, proteins, etc) and guide subsequent in-depth analysis. Overall, this method allows to:
• Cut your specimens in a consistent position and precise manner using microCT-based controlled-angle microtome sectioning.
• Locate and expose a specific anatomical plane (coronal, sagittal plane) or any other anatomical landmarks of interest based on microCT.
• Identify any cell or tissue markers based on IHC to guide further in-depth examination of those regions of interest.
