Processing and Sectioning Undecalcified Murine Bone Specimens

Preparation of mineralized tissue specimens for bone-specific staining encompasses a critical sequence of histological techniques that provides visualization of tissue and cellular morphology. Bone specimens are fixed in 10% neutral buffered formalin (NBF), dehydrated in graded ethanol (EtOH) solutions (and optionally cleared in xylene), infiltrated and embedded in polymethyl methacrylate (methyl methacrylate or MMA), classically sliced into 4-10 micrometer (μm) sections, and stained with bone-specific histological stains such as von Kossa (with either nuclear fast red solution counterstain or MacNeal's tetrachrome counterstain), modified Goldner's trichrome, Alizarin Red S, Safranin O, and tartrate-resistant acid phosphatase (TRAP) stain. Here, we describe the tissue processing of mineralized mouse bones from dissection to staining for histological analysis by light microscopy.