UBC Faculty Research and Publications

Visualisation of Multiple Tight Junctional Complexes in Human Airway Epithelial Cells Buckley, Alysia G; Looi, Kevin; Iosifidis, Thomas; Ling, Kak-Ming; Sutanto, Erika N; Martinovich, Kelly M; Kicic-Starcevich, Elizabeth; Garratt, Luke W; Shaw, Nicole C; Lannigan, Francis J; Larcombe, Alexander N; Zosky, Graeme; Knight, Darryl A; Rigby, Paul J; Kicic, Anthony; Stick, Stephen M Feb 1, 2018

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METHODOLOGY Open AccessVisualisation of Multiple Tight JunctionalComplexes in Human Airway EpithelialCellsAlysia G. Buckley1, Kevin Looi2, Thomas Iosifidis2,3, Kak-Ming Ling4, Erika N. Sutanto4,5, Kelly M. Martinovich4,Elizabeth Kicic-Starcevich4, Luke W. Garratt2, Nicole C. Shaw4, Francis J. Lannigan6, Alexander N. Larcombe4,Graeme Zosky4,7, Darryl A. Knight8,9,10, Paul J. Rigby1, Anthony Kicic2,3,4,5,11,12* and Stephen M. Stick2,3,4,5AbstractBackground: 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.Keywords: Tight junctions, Confocal microscopy, Fixation, Airway epithelial cells, Air liquid interface* Correspondence: anthony.kicic@telethonkids.org.auAlysia G. Buckley and Kevin Looi are co-first authors.Anthony Kicic and Stephen M. Stick are co-senior authors.2School of Paediatrics and Child Health, The University of Western Australia,Nedlands, Western Australia 6009, Australia3Centre for Cell Therapy and Regenerative Medicine, School of Medicine andPharmacology, The University of Western Australia, Nedlands, WesternAustralia 6009, AustraliaFull list of author information is available at the end of the article© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe 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.Buckley et al. Biological Procedures Online  (2018) 20:3 DOI 10.1186/s12575-018-0070-0BackgroundThe airway epithelial layer remains the frontline of defenceagainst pathogens, aeroallergens and noxious gases byestablishing and maintaining a physical barrier. The integ-rity of this layer is typically maintained by the presence ofa range of junctional complexes including: tight junctions;adherens junctions; and desmosomes [1–4]. Apically lo-cated tight junctions perform a fundamental role in regu-lating solute transport across the airway epithelium [5] byrestricting macromolecule migration through paracellularspaces [6–9]. Several families of proteins have been identi-fied to form tight junctions between adjacent cells includ-ing the occludin and claudin families. These proteinscontain four transmembrane domains with two extracellu-lar loops, where the extracellular loops fuse with theircounterpart on adjacent cells [10] resulting in a belt-likestructure around the apical surface of airway epithelial cells[4, 5, 11, 12]. In association with the transmembrane tightjunction proteins is the intracellular protein zonaoccludens-1 (ZO-1) [10] which act by anchoring the tightjunction proteins to the cytoskeleton [13].Studies have observed that decreases in ZO-1, claudin-1and occludin organisation within the cell membrane leadsto disruption of barrier function in epithelial cells, therebyallowing entry of bacteria and other pathogens into thehost [6–9, 14, 15]. Some evidence also suggests that alter-ation of adherens junctions can also facilitate the entry ofpathogens into the host [6, 14]. Recent investigations sug-gest that epithelial barrier integrity may be dysfunctionalin airway diseases such as asthma, where decreased tightjunctional complexes and increased layer permeabilityhave been observed [3, 16, 17]. Tight junction proteinsZO-1 and occludin have also been shown to have lowerexpression and a disorganised structure in asthmatic epi-thelium, when compared to non-asthmatic epithelium,resulting in reduced barrier function [15, 16].Tight junction integrity has typically been assessed usingTransepithelial Electrical Resistance (TEER) [18]. Higherresistance measurements are typically observed in conflu-ent polarised cultures with intact junctional complexessince ions cannot pass across the epithelial cellular layerinto basal compartments [19]. Conversely, low TEER valuesare a consequence of increased ion transport across theepithelial layer, indicative of increased permeability result-ing from incomplete tight junctions [15, 19, 20]. Despitethese measurements providing insight into the globalchanges, they fail to provide insight into localised changesthat may be occurring between cells. Thus, confocalmicroscopy provides a valuable tool for the visualisationand assessment of local protein changes and interactions,and may also be used to corroborate TEER measurements.Here, we optimised and established a methodology forepithelial tissue fixation for the immunocytochemical ana-lysis of tight junctions (ZO-1, claudin-1 and occludin),initially in a representative airway epithelial cell line(16HBE14o-), followed by corroboration in primary airwayepithelial cultures grown at air-liquid interface (ALI).MethodsReagentsThe culture reagents Modified Eagle’s Medium (MEM),Penicillin/Streptomycin, L-Glutamine, Foetal Calf Serum(FCS) and Normal Goat Serum (NGS) were purchasedfrom Life Technologies (CA, USA). Triton X-100, trizmabase, sodium chloride, bovine serum albumin (BSA) andfibronectin were purchased from Sigma Aldrich (MO,USA). Collagen IV was purchased from BD Biosciences(New Jersey, USA).AntibodiesFor immunocytochemistry, the following antibodies wereused: Claudin-1 (polyclonal), Occludin (monoclonal,clone OC-3F10), ZO-1 (monoclonal, clone ZO1-1A12,and polyclonal), AlexaFluor 488 (Goat anti-Mouse andGoat anti-Rabbit) and AlexaFluor 568 (Goat anti-Mouseand Goat anti-Rabbit). These antibodies were purchasedfrom Life Technologies (CA, USA). Hoechst 33,342 waspurchased from Sigma Aldrich (MO, USA).Cell Culture and Maintenance16HBE14o- cells, a SV-40 transformed bronchial epithe-lial cell line, were kindly provided by Dr. Dieter Gruenet(University of California, San Francisco, USA). Cellswere cultured in MEM containing 10% (v/v) FCS,100 U/mL (v/v) Penicillin/Streptomycin and 1% (v/v) L-Glutamine in a 37 °C, 5% CO2 incubator. For experi-ments, cells were seeded at a density of 10,000 cells/coverslip on glass coverslips previously coated with10 μg/mL fibronectin, 30 μg/mL collagen I and 100 μg/mL BSA. Cells were maintained under standard cultureconditions until 100% confluency over the coverslipswas achieved. Cultures were then continued for a further3 days before being fixed for subsequent immunocyto-chemical analysis to ensure complete generation of tightjunction proteins.Establishment of ALI CulturesPrimary airway epithelial cells (AECs) were obtained fromchildren admitted for elective surgery for non-respiratoryrelated conditions [21–23] and de-identified prior to down-stream analysis. Primary AECs were then grown on 6.5-mm Transwell-Clear inserts 0.4 μm pore size (Corning,NY, USA) pre-coated with 30 μg/mL human placentalcollagen type I, which has been previously demonstrated tosupport AEC growth [24]. Cells were grown undersubmerged conditions in Bronchial-Air Liquid Interface(B-ALI™, Lonza, MD, USA) growth media until confluent.To differentiate into ciliated pseudostratified AECs, mediaBuckley et al. Biological Procedures Online  (2018) 20:3 Page 2 of 9was removed from the apical side and this was consideredDay 0 of ALI culture and the start of the experimentalperiod. Cells were then grown in B-ALI™ differentiationmedia, added to the basolateral side every alternate day andthe apical side washed with tissue-culture sterile 1X PBSweekly. Cultures were grown for 28 days at ALI to ensuremaximal differentiation as assessed by the presence of beat-ing cilia as well as mucus production, as evident by mucusbuild-up on the apical side of the cultures.FixationThis study sought to investigate various fixation methodssuitable for the reproducible staining of epithelial airwaycells. All treatments were repeated in triplicate. All fixationcombinations can be found in Table 1.ParaformaldehydeCells were fixed using 4% (v/v) paraformaldehyde in71 mM Tris Buffered Saline (TBS), pH 7.4, at roomtemperature (RT) for 15 min, followed by washing withTBS for 30 min at RT, replacing wash TBS every 5 min.Cells were then stored in TBS at 4 °C until required.Methanol, Acetone, Methanol: AcetoneFixation with coagulant fixatives was performed usingeither ice cold 100% methanol, acetone or 1:1 methanol:acetone. Cells were fixed at − 20 °C for 10 min, followed bywashing with TBS for 30 min at RT, replacing wash TBSevery 5 min. Cells were then stored in TBS at 4 °C untilrequired.Triton X-100 Pre-extractionCells were incubated with 0.2% (v/v) Triton X-100 in 1×TBS on ice for 10 min, followed by gentle washing withTBS for 30 min at RT, replacing wash TBS every 5 min.Fixation following pre-extraction was performed witheither methanol or paraformaldehyde as describedabove. Cells were then stored in TBS at 4 °C untilrequired.PermeabilizationFollowing fixation, permeabilization was performed on anumber of samples. Here, cells were treated with 0.1%(v/v) Saponin in 1× TBS and incubated at RT for10 min. Cells were then washed with 1× TBS for 30 minat RT, replacing wash TBS every 5 min. Cells were thenstored in TBS at 4 °C until required.Blocking SolutionTo minimise non-specific binding of primary and second-ary antibodies in samples, blocking solution containing10% (v/v) NGS, 10% (v/v) FCS and 1% (v/v) BSA in 1×TBS was incubated on cells for 30 min at RT. For parafor-maldehyde fixed samples, 0.2% (v/v) Triton X-100 was in-cluded in the blocking solution. In addition, all antibodieswere diluted in the blocking solution outlined above.ImmunocytochemistryPrimary antibodies were incubated on cells for 1 h atRT, followed by washing with 1× TBS at RT every10 min for 1 h. Secondary antibody incubation and washwas performed as per the primary antibody incubationstep. Cells were also incubated with Hoechst (2.5 μg/mL) for 5 min at RT during the final wash step to stainfor nuclei. All coverslips were mounted with mountingmedium containing 19 mM polyvinyl alcohol (PVA,Sigma Aldrich, MO, USA), 45 mM Trizma Base (SigmaAldrich, MO, USA), 45 mM NaH2PO4.2H2O, 27% (v/v)glycerol (Sigma Aldrich, MO, USA), and 4.9 mM chloro-butanol (Sigma Aldrich, MO, USA). Negative controlsamples were included to determine the level of non-specific binding of secondary antibodies to the tissue.Confocal MicroscopyTreated and control samples were imaged using a NikonA1 inverted confocal microscope (Nikon, Japan), with aNikon Plan Apo VC 60× NA 1.4 oil immersion objective(Nikon, Japan) and NIS-AR Elements software (v4.2.22,Nikon, Japan). Individual channels were captured sequen-tially, where a 405 nm laser was used for Hoechst 33,342with collection through a 450/50 bandpass filter, AF488excited using a 488 nm laser with collection through 525/50, and AF568 excited with a 561 nm laser and collectedthrough a 585/50 bandpass filter. Z-stack images with stepsize of 0.5 μm were collected with a pinhole of 35.8 μm(1.2 A.U. for 488 nm laser), where the top and bottom ofthe stacks were determined visually.Table 1 Fixative combinations used in this study. All fixativecombinations were performed in triplicate on 16HBE14o-cultured cells. Immunocytochemistry was performed on cells asdetailedPre-extraction Fixation Permeabilization– 4% Paraformaldehyde –0.2% Triton X-100 4% Paraformaldehyde –– 4% Paraformaldehyde 0.1% Saponin– 4% Paraformaldehyde+ Acetone–– 100% Methanol –0.2% Triton X-100 100% Methanol –– 100% Methanol 0.1% Saponin– 1:1 Methanol:Acetone –– 100% Acetone –Buckley et al. Biological Procedures Online  (2018) 20:3 Page 3 of 9ResultsTo determine the extent of tight junction formation inepithelial cells we examined the effect of various fixativeson the epithelial cell line 16HBE14o- (Table 2). Initial ex-periments did not produce staining for any fixation combi-nations where fibronectin/collagen coating of coverslipswas omitted (data not shown). Coverslip coating was usedfollowing these preliminary experiments to aid adherenceand cell growth [25]. Image analysis of paraformaldehydefixed cells, with a Triton X-100 permeabilization step,showed no specific staining of tight junction complexesZO-1, occludin or claudin-1 (Fig. 1). Saponin was used fol-lowing paraformaldehyde fixation as an alternativepermeabilization agent to Triton X-100. Data generatedshowed that use of saponin slightly increased junctionalstaining post paraformaldehyde fixation for ZO-1, butpositive staining was highly variable within samples(data not shown). Occludin and claudin-1 stainingwas absent following paraformaldehyde-saponin fix-ation and permeabilization. Pre-extraction with 0.2%Triton X-100 on ice, followed by fixation, was alsotested. However, following the pre-extraction treat-ment, all cells lost attachment to the coverslip andimmunocytochemistry was not performed (data notshown). Using a combinatorial approach of parafor-maldehyde fixation, followed by acetone fixation toincrease permeabilization, also failed to yield positivestaining for ZO-1, occludin or claudin-1 (data notshown).Coagulative fixation methods were also tested to deter-mine epitope accessibility. Positive staining was observedfollowing methanol fixation for ZO-1 and claudin-1 tightjunction proteins, but no staining was detected for occlu-din (Fig. 2). Fixation with acetone failed to expose anypositive staining for ZO-1, occludin or claudin-1 in thesamples (data not shown). Permeabilization with saponinfollowing methanol fixation was unsuccessful in produ-cing tight junction staining for any of the antibodiesassessed (data not shown). Combinatorial coagulative fix-ation produced consistent positive staining for both ZO-1and occludin. However, no claudin-1 staining was ob-served (Fig. 3). No fluorescence was observed in negativecontrols for all fixation methods, where positive samplesettings were used (data not shown).Following optimisation of the staining protocol, stain-ing on primary cells cultured to ALI was performed toverify compatibility between the transformed cell lineand primary cells. Fixation of ALI culture with methanolshowed positive staining for both claudin-1 and ZO-1,as seen with the 16HBE14o- cell line, whilst fixationwith 1:1 methanol: acetone produced positive stainingfor occludin and ZO-1 (Fig. 4).DiscussionIn the current study, we found that fixation of16HBE14o- cells for the tight junction proteins ZO-1,claudin-1 and occludin require different fixation proto-cols for reliable staining patterns. For consistent stainingof claudin-1, fixation with ice cold methanol was re-quired, whilst occludin needed a combinatorial approachof methanol: acetone. We found that staining for ZO-1could be positively identified using both fixation ap-proaches, and as such, could be used as a counter stainfor both claudin-1 and occludin. Furthermore, we foundthat staining patterns in cell line 16HEB14o- was con-gruent with primary airway epithelial cells grown in ALI.This consistency in staining pattern reinforces their use-fulness as a substitute for protocol optimization, as ac-cess to paediatric primary epithelial cells is often limited.Methodologies to perform reproducible immunocyto-chemistry for tight junction proteins (ZO-1, occludin andclaudin-1) in epithelial derived cells to date are inconsist-ent and, at times, conflicting [6, 26, 27]. Particularly forepithelial cells, the fixation method must be carefullychosen to ensure optimal staining for the antigens ofinterest [28, 29]. Routine histological fixatives are oftenused without thought as to why one fixative may be bettersuited for a particular antigenic epitope than another.The most common types of fixatives used in immuno-cytochemistry fall into two categories: (1) non-coagulativeor cross-linking and (2) coagulative fixatives. The cross-linking family include formaldehyde and glutaraldehyde[29]. These fixatives transform the cytosol into an insol-uble gel by the formation of methylene bridges betweenproteins, which halt autolysis and harden tissue [30, 31].Fixation via this method may alter some of the tertiaryTable 2 Qualitative assessment of fluorescent staining of tightjunction antibodies, where: - indicates negative staining; +indicates weak staining with no consistent structure; ++indicates moderate staining of tight junctions, with somestructure present; +++ indicates strong staining, with consistentstructures presentFixation Claudin-1 Occludin ZO-14% Paraformaldehyde – – –0.2% Triton Pre-extraction+ 4% Paraformaldehyde– – –4% Paraformaldehyde+ 0.1% Saponin– – +4% Paraformaldehyde+ Acetone– – –Methanol +++ – +++0.2% Triton Pre-extraction+ Methanol– – –Methanol + 0.1% Saponin + – +Methanol + Acetone – +++ +++Acetone – – –Buckley et al. Biological Procedures Online  (2018) 20:3 Page 4 of 9protein structure within the tissue, but generally maintainssecondary protein structures [29, 31–33]. Absence ofclaudin-1 and occludin antibody labelling using this fix-ation method in our laboratory may be due to the locationof the proteins within the cell membrane, preventing suffi-cient access of the antibody to the epitope. Whilst theZO-1 protein is not located within the cellular membrane,the negative staining following paraformaldehyde fixationmay also be attributable to the restricted epitope accessdue to cross-linked adjacent proteins, or steric hindrance.As the cross-linking fixatives change cytoplasm intoan insoluble gel, permeabilization steps may be requiredfor immunocytochemical analysis of intracellular com-ponents [32, 34, 35]. Surfactants and non-ionic deter-gents, such as saponin and Triton X-100 respectively,are commonly used in immunocytochemistry for theFig. 1 Paraformaldehyde fixation (4%) of 16HBE14o- cells in culture. The top row of panels show absence of specific staining for claudin-1 (Green)and ZO-1 (Red). The bottom row of panels show co-staining of occludin with ZO-1. Merged images showed nuclei staining with Hoechst (blue)Fig. 2 Methanol fixation of 16HBE14o- cells in culture. The top row of panels show claudin-1 co-stained with ZO-1. The bottom row of panelsshow the destruction of occludin staining using methanol as the fixative. Merged images showed nuclei staining with Hoechst (blue)Buckley et al. Biological Procedures Online  (2018) 20:3 Page 5 of 9purpose of increasing cellular permeability [35–38]. Solu-bilisation of lipid components non-specifically by TritonX-100, or specific cholesterol removal by saponin, facili-tates antibody access to intracellular compartments andepitopes without changing the cells’ ultrastructural integ-rity [28]. Saponin might be expected to increase epitopeexposure for ZO-1, occludin or claudin-1. However, inour samples, permeabilization with saponin did not alterantibody staining of occludin or claudin-1 tight junctionproteins, with occasional variable staining for ZO-1. Thisnegligible staining following the use of surfactants may bedue to the epitope for claudin-1 and occludin beinglocated in a position that is not altered by the removal of,nor coupled to, lipids or cholesterol.Fig. 3 Methanol-acetone (1:1) fixation of 16HBE14o- cells in culture. The top row of panels show the absence of claudin-1 staining, whilst ZO-1staining is clearly visible. The bottom row of panels show co-staining of occludin with ZO-1 using methanol-acetone as the fixative. Merged im-ages showed nuclei staining with Hoechst (blue)Fig. 4 Methanol-acetone fixation of primary airway epithelial cells (AEC) grown at air liquid interface (ALI). The top row of panels show positivestaining for both claudin-1 and ZO-1. The bottom row of panels show positive co-staining of occludin and ZO-1 in primary AECs grown at ALI.Merged images showed nuclei staining with Hoechst (blue)Buckley et al. Biological Procedures Online  (2018) 20:3 Page 6 of 9Pre-extraction with Triton X-100, followed by fixationwith paraformaldehyde, has been suggested to removesome background staining in tissues, as some solublecomponents within the cell are removed prior to fixation[29, 34]. As such, this should provide greater access forantibodies to bind to epitopes of interest, as the lipids areremoved in a non-selective manner. However, followingexposure of confluent 16HBE14o- cells to Triton X-100,all cells appeared to lose attachment to the extracellularmatrix (ECM). There are also suggestions that paraformal-dehyde is unable to sufficiently cross-link proteins in situ[39], although other studies suggest that formaldehyde isonly released from tissues following years of washing tis-sues in water, and cross-linking bonds cannot be brokenby urea [40]. In our study, it is likely that the epitopes ofinterest are insufficiently exposed via the cross-linking fix-ation and permeabilization methods commonly employed.Coagulant fixatives are also commonly used to fix tissuefor immunocytochemistry. This family includes alcoholssuch as ethanol and methanol, as well as acetone [33]. Fix-ation of our samples with coagulant fixatives producedvaried results. The use of methanol fixation revealed posi-tive staining for ZO-1, but occludin staining was absent.Alcohol fixatives simultaneously fix and permeabilize cells,by extracting phospholipids and precipitating proteins intissue [41]. They are frequently used for observing cellularcytoskeletal elements, as shown with the positive ZO-1tight junction staining. The coagulant fixatives displacewater molecules from proteinaceous materials, therebybreaking hydrogen bonds [42]. Alterations of hydrogenbonds can change the tertiary structure of proteins butdoes not alter the amino acid sequence of the epitope[42]. This can result in exposure of epitopes which werepreviously buried within the protein, thereby allowingantibody access and binding [42]. This alteration of pro-tein tertiary structure protein may not have been sufficientto unmask the occludin epitope, and as such, further in-vestigation was required.Acetone is another coagulative fixative with strong lipidremoval activity, particularly triglycerides and sterols [43].In our samples, fixation with acetone failed to producepositive staining for any tight junction proteins. As acetoneis a stronger organic solvent than alcohol, cell membraneloss can be observed following cellular fixation [31, 43]. Tochange the epitope exposure, without complete loss of cel-lular membranes, a fixative of 1:1 methanol: acetone wasperformed. Staining following dual fixation showed positivefluorescence for ZO-1 and occludin proteins, howeverclaudin-1 staining was destroyed. It is plausible that theextra denaturation required for occludin antibody accessresults in masking of the claudin-1 epitope.The optimized fixation protocol was then repeated onprimary airway epithelial cell culture samples derived fromhealthy participants, where cells had successfully reacheda differentiated state when grown under ALI conditions.Fixation of the cultures with methanol yielded positivestaining with claudin-1 and ZO-1, whilst methanol: acet-one fixation yielded positive staining for occludin and ZO-1, reproducing the findings seen with the 16HBE14o− cul-tures. However, it should be noted that there were subtledifferences in the staining intensity as well as the patternof staining, suggesting that the final interpretation ofstaining should be restricted to primary cultures and notwith the surrogate optimisation model.ConclusionsIn conclusion, this study successfully established a meth-odological workflow (Fig. 5) using confocal microscopy tocompare and evaluate staining expression levels of mul-tiple tight junction complexes in the human airway. Viathe workflow, we established that there was no universalmethodological approach appropriate for staining andvisualising all tight junction proteins investigated. How-ever, we identified key points within the methodologicalworkflow which after specialised optimisation lead to sub-sequent visualisation of each tight junction protein.Finally, we successfully demonstrated the reproducibilityand translation of the workflow in primary AEC ALI cul-tures, indicating the adaptability of this method in othercell types. Of significance, this workflow can now be usedFig. 5 Schematic representation of the workflow required for the visualization of tight junctional complexes in airway epithelial cells. * denoteskey points within the workflow which requires specialized optimizationBuckley et al. Biological Procedures Online  (2018) 20:3 Page 7 of 9to visualise epithelial tight junctions and assess barrier in-tegrity in established cell cultures derived from chronicairway diseases including cystic fibrosis, chronic obstruct-ive pulmonary disorder and asthma.AbbreviationsAEC: Airway epithelial cell; ALI: Air-liquid interface; B-ALI: Bronchial Air-LiquidInterface; BSA: Bovine Serum Albumin; FCS: Foetal Calf Serum;MEM: Minimum Essential Medium; NGS: Normal Goat Serum; pAECs: Primaryairway epithelial cells; PBS: Phosphate Buffered Saline; PVA: Polyvinyl Alcohol;RT: Room Temperature; TBS: Tris Buffered Saline; TEER: TransepithelialElectrical Resistance; ZO-1: Zona Occludens-1AcknowledgementsThe authors acknowledge the facilities, and the scientific and technicalassistance of the Australian Microscopy & Microanalysis Research Facility atthe Centre for Microscopy, Characterisation & Analysis, The University ofWestern Australia, a facility funded by the University, State andCommonwealth Governments. We would like to thank the contribution andassistance of all the respiratory fellows, anesthetists, nurses and hospital staffat Princess Margaret Hospital and St John of God. Finally, we would also liketo thank the families and children participating in this study.FundingThis work was supported by grants from the National Health and MedicalResearch Council of Australia (1026494 & 1048910). Stephen M. Stick is aNHMRC Practitioner Fellow.Availability of Data and MaterialsAll data generated or analysed during this study are included in thispublished article and are available from the corresponding author onreasonable request.Authors’ ContributionsAGB performed all the optimisation experiments in the cell line, conductedall experiments in the primary cell cultures and drafted the manuscript. KL,assisted with initial sample processing, cell culture establishment,maintenance of the primary cell cultures grown at air liquid interface andcritically revised the manuscript. KML, ENS, KMM, EKS, LWG, NCS, all assistedwith the human sample recruitment, sample processing, cell cultureestablishment and maintenance. FL performed the airway sampling andcritically revised the manuscript. ANL, GZ, DAK, PJR and SMS were involvedin the concept design and coordination of the study and critically revisedthe manuscript. AK optimized and established the protocols for cell culture,assisted with the concept and design of the study, assisted in samplecollection and processing and critically revised the manuscript. All authorshave read and approved the manuscript.Ethics Approval and Consent to ParticipateThis study was approved by the Human Ethics Committee for both PrincessMargaret Hospital for Children (Reference Number 1402EP, 1903EP) and StJohn of God Hospital (Reference Number 452).Consent for PublicationNot applicable.Competing InterestsThe authors declare that they have no competing interests.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.Author details1Centre of Microscopy, Characterisation and Analysis, The University ofWestern Australia, Crawley, Western Australia 6009, Australia. 2School ofPaediatrics and Child Health, The University of Western Australia, Nedlands,Western Australia 6009, Australia. 3Centre for Cell Therapy and RegenerativeMedicine, School of Medicine and Pharmacology, The University of WesternAustralia, Nedlands, Western Australia 6009, Australia. 4Telethon Kids Institute,Centre for Health Research, The University of Western Australia, Crawley,Western Australia 6009, Australia. 5Department of Respiratory Medicine,Princess Margaret Hospital for Children, Perth, Western Australia 6001,Australia. 6School of Medicine, Notre Dame University, Fremantle, WesternAustralia 6160, Australia. 7School of Medicine, Faculty of Health, University ofTasmania, Hobart, Tasmania 7000, Australia. 8School of Biomedical Sciencesand Pharmacy, University of Newcastle, Callaghan, New South Wales,Australia. 9Priority Research Centre for Asthma and Respiratory Disease,Hunter Medical Research Institute, Newcastle, New South Wales, Australia.10Department of Anesthesiology, Pharmacology and Therapeutics, Universityof British Columbia, Vancouver, Canada. 11School of Public Health, CurtinUniversity, Bentley, Western Australia 6102, Australia. 12Telethon Kids Institute,Subiaco, Perth, Western Australia 6008, Australia.Received: 25 July 2017 Accepted: 22 January 2018References1. 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