Osteoclast visualization: Tartrate-resistant acid phosphatase activity staining using NewFuchsin compatible with non-aqueous mounting and tissue clearing

IF 1.6 Q2 MULTIDISCIPLINARY SCIENCES

MethodsX Pub Date : 2024-12-26 DOI:10.1016/j.mex.2024.103136

Takashi Nakamura , Katsuhiro Kawaai , Yukiko Kuroda , Koichi Matsuo

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Abstract

Tartrate-resistant acid phosphatase (TRAP) staining is widely used to stain osteoclasts in histological bone sections. The red dye formed by the conventional TRAP enzymatic reaction using naphthol AS-MX (or AS-BI) phosphate and fast red-violet (or garnet) chromogens is readily soluble in alcohol or xylene and requires air-drying prior to cover slipping or the use of an aqueous mounting medium. However, the use of an aqueous mounting medium makes it difficult to store stained specimens for a long time. In this modified method, a new fuchsin (NewFuchsin) was used as a chromogen, which enabled dehydration and clearing after staining and the use of a non-aqueous organic solvent-based mounting medium. Samples prepared using this modified TRAP activity staining method (NewFuchsin TRAP staining) have the following advantages over conventional TRAP staining:

•
The staining of sections provides a clear histological image and allows for long-term preservation.
•
The red dye formed by NewFuchsin TRAP staining can be detected not only in the bright field, but also in the fluorescent field.
•
Combined with tissue clearing using ethyl cinnamate, osteoclasts are observed using three-dimensional imaging.

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破骨细胞可视化：使用与非水固定和组织清除相容的新品红进行抗酒石酸酸性磷酸酶活性染色。

抗酒石酸酸性磷酸酶（TRAP）染色广泛用于骨组织学切片的破骨细胞染色。使用萘酚AS-MX（或AS-BI）磷酸盐和快速红紫色（或石榴石）色素进行常规TRAP酶促反应形成的红色染料很容易溶解于酒精或二甲苯中，在盖层脱落或使用水性安装介质之前需要风干。然而，使用水性安装介质使得染色标本难以长时间保存。在这种改进的方法中，使用一种新的品红（NewFuchsin）作为显色剂，染色后可以脱水和清除，并使用非水有机溶剂基安装介质。使用这种改进的TRAP活性染色方法（NewFuchsin TRAP染色）制备的样品与传统的TRAP染色相比具有以下优点：•切片染色提供清晰的组织学图像，并允许长期保存。•NewFuchsin TRAP染色形成的红色染料不仅可以在明亮场中检测到，也可以在荧光场中检测到。•结合使用肉桂酸乙酯清除组织，使用三维成像观察破骨细胞。

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