ABSTRACT The technique for clearing and staining whole specimens consists of many steps. This study discusses the alcian blue/alizarin red S staining method and aims to provide a useful reference and review for users who intend to do this staining. To specifically address the influences of tissue removal on staining results, the mouse fetuses at embryonic stage E18.5 and adult mice at 12 weeks of age were used in this study. The fetuses were divided into three groups: Group 1 skin, muscle, and viscera removed, Group 2 skin and muscle removed, and Group 3 viscera removed. For successful skeletal staining, it was concluded that (1) skin removal from fetuses was necessary for alcian blue staining but unnecessary for alizarin red S staining, (2) removal of muscle surrounding thorax and neck of fetuses could improve transparency effects, (3) retaining fetal viscera would not significantly affect transparency but might avoid the tissue damage, and (4) complete skin, muscle, and viscera removal were essential for good staining of adult mice. The representative images and detailed staining procedures might be good for researchers presently using alcian blue and alizarin red S staining to differentiate cartilages and ossified bones.

中文翻译：

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使用阿尔新蓝和茜素红 S 对胎儿和成年小鼠骨骼中的软骨和骨化骨进行差异染色的方案

摘要 清除和染色整个标本的技术包括许多步骤。本研究讨论了阿尔新蓝/茜素红 S 染色方法，旨在为打算进行这种染色的用户提供有用的参考和回顾。为了专门解决组织去除对染色结果的影响，本研究使用胚胎期 E18.5 的小鼠胎儿和 12 周龄的成年小鼠。胎儿被分为三组：第一组皮肤、肌肉和内脏被去除，第二组皮肤和肌肉被去除，以及第三组内脏被去除。对于成功的骨骼染色，得出结论：（1）阿尔新蓝染色需要去除胎儿皮肤，而茜素红 S 染色不需要，（2）去除胎儿胸部和颈部周围的肌肉可以提高透明度效果，(3) 保留胎儿内脏不会显着影响透明度但可以避免组织损伤, (4) 完整的皮肤、肌肉和内脏去除对于成年小鼠的良好染色至关重要。代表性图像和详细的染色程序可能对目前使用阿尔新蓝和茜素红 S 染色来区分软骨和骨化骨骼的研究人员有好处。