Triple labelling of actin filaments, intermediate filaments and microtubules for broad application in cell biology: uncovering the cytoskeletal composition in tunneling nanotubes

  • Nataša Resnik
  • Andreja Erman
  • Peter Veranič
  • Mateja Erdani KreftEmail author
Short Communication


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.


Tunneling nanotubes Urothelial cells Urinary bladder tissue Actin filaments Intermediate filaments Microtubules 



Actin filaments


Bovine serum albumin




Intermediate filaments




Phosphate-buffered saline


Tunneling nanotubes



This research work was supported by the Slovenian Research Agency (ARRS) (Grant No. J3-7494 and P3-0108) and MRIC UL IP-0510 Infrastructure program. We express gratitude to Sanja Čabraja, Linda Štrus, Nada Pavlica Dubarič and Sabina Železnik for their technical assistance.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of Cell Biology, Faculty of MedicineUniversity of LjubljanaLjubljanaSlovenia

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