Optimization of Methylmethacrylate Infiltration Duration in Undecalcified Murine Bone Specimens

To obtain high-quality, undecalcified bone sections, specimens must be properly fixed, dehydrated, cleared, infiltrated, embedded, sectioned, and stained. Here, we examine different infiltration durations for methylmethacrylate-embedded mouse tibiae and lumbar vertebrae. Our data indicate that 24 h of vacuum infiltration allows for good sections in both lumbar vertebrae and tibiae, with minimal microtears. Furthermore, we show that with 24 h of infiltration, excellent results were obtained for all seven histochemical and immunohistochemical stains performed. In general, good results were obtained when specimens were infiltrated for 12-72 h. However, with 120 h of infiltration, the marrow retained methylmethacylate even after the sections were deplasticized. Also, when specimens were infiltrated for 6 h, they were underinfiltrated, resulting in increased numbers of microfractures in trabecular bone spicules. Therefore, in today's busy laboratory setting, in which quickly examining a bone phenotype, performing histomorphometry, or special bone-specific stains is of importance, we recommend that mouse tibiae and lumbar vertebrae be infiltrated under vacuum for 24 h at 4 degrees C with methylmethacrylate before embedding. This technique provides a good balance between section/staining quality and time.