Troiano Nancy W, Ciovacco Wendy A, Kacena Melissa A
Department of Orthopaedics and Rehabilitation, Yale University School of Medicine, New Haven, CT.
J Histotechnol. 2009 Mar 1;32(1):27-31. doi: 10.1179/his.2009.32.1.27.
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.
组织学评估是一个复杂的多步骤过程，最终以组织染色告终。在染色之前的所有步骤都会影响最终质量，但这些预处理的影响往往没有得到足够的重视。特别是固定剂的选择通常不是基于其有效性，而是基于便利性和可得性。本研究试图为骨组织组织学评估选择固定剂制定指导原则。我们比较了两种最常用的固定剂——乙醇和福尔马林，它们用于包埋在甲基丙烯酸甲酯中的小鼠胫骨，随后用甲苯胺蓝、番红O或冯·科萨染色。我们的结果表明，与采用相同处理和包埋技术的10%中性缓冲福尔马林固定相比，70%乙醇固定以及随后在甲基丙烯酸甲酯中进行处理，对骨细胞相关成分的染色效果更好。此外，我们证明，额外的丙酮脱水和透明步骤能使70%乙醇固定的骨标本获得更好的可视化效果。然而，额外的丙酮步骤并没有增强10%中性缓冲福尔马林固定的骨标本的可视化效果。最后，与乙醇固定相比，福尔马林固定时骨髓成分更容易可视化。
