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Visualisation of Multiple Tight Junctional Complexes in Human Airway Epithelial Cells

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Buckley, AG, Looi, K, Iosifidis, T, Ling, KM, Sutanto, EN, Martinovich, KM, Kicic-Starcevich, E, Garratt, LW, Shaw, NC, Lannigan, FJ, Larcombe, AN, Zosky, G ORCID: 0000-0001-9039-0302, Knight, DA, Rigby, PJ, Kicic, A and Stick, SM 2018 , 'Visualisation of Multiple Tight Junctional Complexes in Human Airway Epithelial Cells' , Biological Procedures Online, vol. 20, no. 1 , pp. 1-9 , doi: 10.1186/s12575-018-0070-0.

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Abstract

Background: Apically located tight junctions in airway epithelium perform a fundamental role in controllingmacromolecule migration through paracellular spaces. Alterations in their expression may lead to disruptions inbarrier integrity, which subsequently facilitates entry of potential bacterial and other pathogens into the host.Furthermore, there is emerging evidence that the barrier integrity of the airway in certain airway inflammatorydiseases may be altered. However, there is little consensus on the way this is assessed and measured and thetype of cells used to achieve this.Methods: Here, we assessed four fixation methods including; (i) 4% (v/v) paraformaldehyde; (ii) 100% methanol;(iii) acetone or; (iv) 1:1 methanol: acetone. Pre-extraction with Triton X-100 was also performed and assessed oncells prior to fixation with either methanol or paraformaldehyde. Cells were also permeabilized with 0.1% (v/v)Saponin in 1× TBS following fixation and subsequently stained for tight junction proteins. Confocal microscopywas then used to visualise, compare and evaluate staining intensity of the tight junctional complexes in order todetermine a standardised workflow of reproducible staining.Results: Positive staining was observed following methanol fixation for claudin-1 and ZO-1 tight junction proteins butno staining was detected for occludin in 16HBE14o- cells. Combinatorial fixation with methanol and acetone alsoproduced consistent positive staining for both occludin and ZO-1 tight junction proteins in these cells. When assessedusing primary cells cultured at air-liquid interface, similar positive staining for claudin-1 and ZO-1 was observed followingmethanol fixation, while similar positive staining for occludin and ZO-1 was observed following the same combinatorialfixation with methanol and acetone.Conclusions: The present study demonstrates the importance of a personalised approach to optimise staining forthe visualisation of different tight junction proteins. Of significance, the workflow, once optimised, can readily betranslated into primary airway epithelial cell air-liquid interface cultures where it can be used to assess barrierintegrity in chronic lung diseases.

Item Type: Article
Authors/Creators:Buckley, AG and Looi, K and Iosifidis, T and Ling, KM and Sutanto, EN and Martinovich, KM and Kicic-Starcevich, E and Garratt, LW and Shaw, NC and Lannigan, FJ and Larcombe, AN and Zosky, G and Knight, DA and Rigby, PJ and Kicic, A and Stick, SM
Keywords: Methods, tight junction, respiratory epithelial cells
Journal or Publication Title: Biological Procedures Online
Publisher: BioMed Central
ISSN: 1480-9222
DOI / ID Number: 10.1186/s12575-018-0070-0
Copyright Information:

Copyright 2018 The Authors. Licensed under Creative Commons Attribution 4.0 International (CC BY 4.0) https://creativecommons.org/licenses/by/4.0/

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