Instituto Maimónides de Investigación Biomédica de Cordoba (IMIBIC), Cordoba, Spain.
Department of Cell Biology, Physiology and Immunology, Faculty of Medicine, University of Cordoba, Avda. Menéndez Pidal s/n. 14004, Cordoba, Spain.
Sci Rep. 2020 Oct 7;10(1):16659. doi: 10.1038/s41598-020-74031-x.
Morphometry and histology are essential approaches for investigation and diagnosis of musculo-skeletal disorders. Despite the advent of revolutionary methods of image analysis and high resolution three-dimensional imaging technology, basic conventional light microscopy still provides an incisive overview of the structure and tissue dynamics of the musculoskeletal system. This is crucial to both preclinical and clinical research, since several clinically relevant processes, such as bone repair, osteoarthritis, and metabolic bone diseases, display distinct, if not pathognomonic, histological features. Due to the particular characteristics of the skeletal tissues (i.e., the existence of mineralized extracellular matrices), a large number of staining methods applicable to either decalcified or undecalcified tissues are available. However, it is usually the case that several staining methods need to be sequentially applied in order to achieve the different endpoints required to fully assess skeletal tissue structure and dynamics, and to allow morphometric quantification. We describe herein a novel staining method, the RGB trichrome, amenable for application to decalcified, paraffin embedded human musculoskeletal tissues. The acronym RGB corresponds to the three primary dyes used: picrosirius Red, fast Green, and alcian Blue. Although these individual pigments are commonly used either isolated, in binary combinations, or as part of more complex polychrome staining methods, when merged in the RGB trichrome staining produce high-quality/high-contrast images, permitting not only clear identification of different tissues (i.e., the different types of cartilage, bone and fibrous connective tissue), but also discrimination between calcified and uncalcified bone and cartilage, as well as an unexpected diversity of shades of color, while displaying singular properties among polychrome staining methods, such as the unveiling of the bone osteocyte dendritic/canalicular network. Hence, we propose the RGB trichrome as simple but highly-reliable tool for the preclinical and clinical study of the musculoskeletal system.
形态计量学和组织学是研究和诊断肌肉骨骼疾病的重要方法。尽管出现了革命性的图像分析方法和高分辨率三维成像技术，但基本的常规光镜仍然提供了对肌肉骨骼系统结构和组织动态的深刻概述。这对于临床前和临床研究都至关重要，因为几种与临床相关的过程，如骨修复、骨关节炎和代谢性骨疾病，显示出独特的、如果不是特征性的组织学特征。由于骨骼组织的特殊特性（即矿化细胞外基质的存在），有大量适用于脱钙或未脱钙组织的染色方法。然而，通常情况下，需要顺序应用多种染色方法，以达到充分评估骨骼组织结构和动态所需的不同终点，并允许形态计量学定量。我们在此描述了一种新的染色方法，即 RGB 三色染色法，适用于脱钙、石蜡包埋的人类肌肉骨骼组织。缩写 RGB 对应于使用的三种主要染料：苦味酸红、固绿和阿利新蓝。虽然这些单独的颜料通常单独使用，或者以二进制组合使用，或者作为更复杂的多色染色方法的一部分使用，但在 RGB 三色染色中合并使用时会产生高质量/高对比度的图像，不仅可以清楚地识别不同的组织（即不同类型的软骨、骨和纤维结缔组织），还可以区分钙化和未钙化的骨和软骨，以及意想不到的颜色多样性，同时显示出多色染色方法之间的独特特性，例如揭示骨骨细胞树突/管腔网络。因此，我们建议 RGB 三色染色法作为研究肌肉骨骼系统的临床前和临床研究的简单但高度可靠的工具。
