The Effects of Fixation and Dehydration on the Histological Quality of Undecalcified Murine Bone Specimens Embedded in Methylmethacrylate

Abstract

Histological evaluation is a complex, multistep process culminating in tissue staining. All of the steps leading up to the staining affect the final quality, but too often the effects of these preparations are not given enough consideration. Fixatives in particular usually are chosen not for efficacy but for convenience and availability. This study attempts to create guidelines for selecting fixatives for bone tissue histological evaluation. We compared two of the most widely used fixatives, ethanol and formalin, in their use on mouse tibias embedded in methylmethacrylate and subsequently stained with toluidine blue, safranin O, or Von Kossa. Our results show that ethanol fixation (70%) and subsequent processing in methylmethacrylate gives better staining results for bone cell related elements than fixing in 10% neutral buffered formalin with the same processing and embedding techniques. Further we demonstrated than an additional acetone dehydration and clearing step allowed for even better visualization in bone specimens fixed with 70% ethanol. However, the additional acetone step did not enhance visualization in bone specimens fixed with 10% neutral buffered formalin. Finally, marrow elements were more easily visualized when fixed with formalin as opposed to ethanol.