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, Visualisation of multiple tight junctional complexes in human airway epithelial cells, Biological Procedures Online, 20, (1) Article 3. ISSN 1480-9222 (2018) [Refereed Article]
Copyright 2018 The Authors. Licensed under Creative Commons Attribution 4.0 International (CC BY 4.0) https://creativecommons.org/licenses/by/4.0/
Background: Apically located tight junctions in airway epithelium perform a fundamental role in controlling macromolecule migration through paracellular spaces. Alterations in their expression may lead to disruptions in barrier 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 inflammatory diseases may be altered. However, there is little consensus on the way this is assessed and measured and the type 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 on cells 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 microscopy was then used to visualise, compare and evaluate staining intensity of the tight junctional complexes in order to determine a standardised workflow of reproducible staining.
Results: Positive staining was observed following methanol fixation for claudin-1 and ZO-1 tight junction proteins but no staining was detected for occludin in 16HBE14o- cells. Combinatorial fixation with methanol and acetone also produced consistent positive staining for both occludin and ZO-1 tight junction proteins in these cells. When assessed using primary cells cultured at air-liquid interface, similar positive staining for claudin-1 and ZO-1 was observed following methanol fixation, while similar positive staining for occludin and ZO-1 was observed following the same combinatorial fixation with methanol and acetone.
Conclusions: The present study demonstrates the importance of a personalised approach to optimise staining for the visualisation of different tight junction proteins. Of significance, the workflow, once optimised, can readily be translated into primary airway epithelial cell air-liquid interface cultures where it can be used to assess barrier integrity in chronic lung diseases.
|Item Type:||Refereed Article|
|Keywords:||methods, tight junction, respiratory epithelial cells, confocal microscopy, fixation, air liquid interface|
|Research Division:||Biomedical and Clinical Sciences|
|Research Group:||Cardiovascular medicine and haematology|
|Research Field:||Respiratory diseases|
|Objective Group:||Clinical health|
|Objective Field:||Clinical health not elsewhere classified|
|UTAS Author:||Zosky, G (Professor Graeme Zosky)|
|Web of Science® Times Cited:||10|
|Downloads:||65 View Download Statistics|
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