Department of Cell Biology, Histology and Pharmacology, School of Medicine and INCyL, University of Valladolid, Spain.
Neurosurgery, University Clinical Hospital of Valladolid, Spain.
Micron. 2020 May;132:102841. doi: 10.1016/j.micron.2020.102841. Epub 2020 Jan 25.
The histological study of hard pieces such as tendons and calcified lesions and tissues is a field that has been gaining increased attention owing to the rapid development of implantable prostheses, among other factors. In these studies, serial sectioning is utilized to detect areas of interest throughout the entire piece, as it enables the application of the appropriate light and electron microscopy techniques in these areas. We propose the "three-sectioning method" that subjects the pieces to three consecutive cycles of embedding and sectioning to localize and study the areas of interest, as an efficient technique for these histological studies. The pieces were first embedded in epoxy resin and then cut into thick sections (approximately 300 μm) for the first cycle. Next, areas of interest selected on these thick sections were re-embedded in epoxy resin to be sectioned again (second sectioning) to obtain a series of semithin sections (1-3 μm). These semithin sections are usually studied using the most relevant techniques for light microscopy. Smaller areas of interest are selected to be cut into ultrathin sections (60-90 nm) for transmission electron microscopy. If necessary, the selected areas of the semithin sections can be embedded again, and then cut into new ultrathin sections. The different kinds of sections we have described here may also be studied using scanning electron microscopy. This systematic method facilitates correlative microscopy from lower to higher magnifications along with the usage of a broad variety of histological techniques including electron microscopy.
由于植入式假体等因素的快速发展，对肌腱和钙化病变等硬性组织以及组织的组织学研究受到了越来越多的关注。在这些研究中，通过连续切片来检测整个标本中感兴趣的区域，因为这可以在这些区域应用适当的光学和电子显微镜技术。我们提出了“三切片法”，将标本经过三个连续的包埋和切片循环，以定位和研究感兴趣的区域，这是一种用于这些组织学研究的有效技术。标本首先用环氧树脂包埋，然后切成第一循环的厚切片（约 300μm）。接下来，在这些厚切片上选择感兴趣的区域，再次用环氧树脂重新包埋，再次切片（第二次切片）以获得一系列半薄切片（1-3μm）。这些半薄切片通常使用最相关的光学显微镜技术进行研究。选择较小的感兴趣区域切成超薄切片（60-90nm）用于透射电子显微镜。如果需要，可再次对选定的半薄切片区域进行包埋，然后切成新的超薄切片。我们在这里描述的不同类型的切片也可以使用扫描电子显微镜进行研究。这种系统的方法可以从低倍放大到高倍放大进行相关显微镜检查，并结合使用多种组织学技术，包括电子显微镜。
