An Optimized Methyl Methacrylate Embedding Protocol for Undecalcified Bone Histology Applications in Skeletal Biology

Methyl methacrylate (MMA) impregnation and embedding procedures have been extensively employed for the examination of bone tissue to visualize microstructural and cellular details for quantifiable histological analyses. Current detailed MMA embedding protocols may require expensive instruments or limit use with certain tissue preparations (e.g., fresh). These techniques often require excessive heat, various chemicals for fixation and dehydrating, long fixation times, or decalcification. The introduction of such variables may result in damage to fragile and invaluable bone samples. Our newly developed protocol introduces a time-efficient MMA embedding technique allowing for replicable results for bone sections as thin as 50-100 μm from samples from diverse conditions (e.g., fresh, embalmed, diagenetic) and various animals (e.g., human, cervids, swine, lagomorphs). The presented technique limits heat and chemical exposure, does not require decalcification, reduces the amount of bone required, and significantly decreases embedding time. Over 300 trials were performed to optimize the procedure to ensure replicability. Our embedding protocol is currently being employed for the histological preparation of bone specimens from a large-scale modern human skeletal collection, the Andronowski Skeletal Collection for Histological and Imaging Research. The embedding procedure presented here will further extend the long-term fixation and preservation of samples for microscopy and imaging applications beyond traditional epoxy resin and hardener mounting systems.