- Title
- Visualisation of Multiple Tight Junctional Complexes in Human Airway Epithelial Cells
- Creator
- Buckley, Alysia G.; Looi, Kevin; Larcombe, Alexander N.; Zosky, Graeme; Knight, Darryl A.; Rigby, Paul J.; Kicic, Anthony; Stick, Stephen M.; Iosifidis, Thomas; Ling, Kak-Ming; Sutanto, Erika N.; Martinovich, Kelly M.; Kicic-Starcevich, Elizabeth; Garratt, Luke W.; Shaw, Nicole C.; Lannigan, Francis J.
- Relation
- Biological Procedures Online Vol. 20, Issue 1, no. 3
- Publisher Link
- http://dx.doi.org/10.1186/s12575-018-0070-0
- Publisher
- Biomed Central (BMC)
- Resource Type
- journal article
- Date
- 2018
- Description
- 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 x 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.
- Subject
- tight junctions; confocal mircoscopy; fixation; airway epithelial cells; air liquid interface; SDG 3; Sustainable Development Goals
- Identifier
- http://hdl.handle.net/1959.13/1466207
- Identifier
- uon:47470
- Identifier
- ISSN:1480-9222
- Rights
- This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Language
- eng
- Full Text
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