We report a protocol for simultaneous triple labelling of intermediate filaments, microtubules and actin filaments. The described procedure offers an optimal preservation of the structure and antigenicity of individual representatives of cytoskeletal elements and is applicable for labelling of tissue samples and cultured cells. Namely, we demonstrate that using this protocol the cytoskeletal elements are well-preserved and detectable in the whole mount urinary bladder tissue pieces, cryosections of the urinary bladder, and in cultured normal and cancer urothelial cells including their delicate intercellular connections such as tunneling nanotubes (TnTs). The protocol uncovers for the first time the co-distribution of actin filaments, intermediate filaments and microtubules in TnTs, which were up to now known as mono- or bi-cytoskeletal structures. Presented triple labelling protocol provides an efficient tool for studying co-distribution of actin filaments, intermediate filaments, and microtubules and therefore offers new insights into their cellular and tissue distribution.

中文翻译：

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肌动蛋白丝，中间丝和微管的三重标记在细胞生物学中得到广泛应用：揭示隧穿纳米管中的细胞骨架成分。

我们报告的协议同时中间丝，微管和肌动蛋白丝的三重标记。所描述的程序提供了细胞骨架元素代表的结构和抗原性的最佳保存，可用于组织样品和培养细胞的标记。也就是说，我们证明了使用此协议，在整个坐骑的膀胱组织块，膀胱冰冻切片以及培养的正常和癌细胞尿道上皮细胞（包括其精细的细胞间连接，例如隧道式碳纳米管）中，细胞骨架元素都得到了很好的保存和检测。 TnTs）。该协议首次揭示了肌动蛋白丝，中间丝和微管在TnT中的共分布，这些分布迄今为止被称为单细胞或双细胞骨架结构。