title: Critical importance of appropriate fixation conditions for faithful imaging of receptor microclusters.
abstract: Receptor clustering is known to trigger signalling events that contribute to critical changes in cellular functions. Faithful imaging of such clusters by means of fluorescence microscopy relies on the application of adequate cell fixation methods prior to immunolabelling in order to avoid artefactual redistribution by the antibodies themselves. Previous work has highlighted the inadequacy of fixation with paraformaldehyde (PFA) alone for efficient immobilisation of membrane-associated molecules, and the advantages of fixation with PFA in combination with glutaraldehyde (GA). Using fluorescence microscopy, we here highlight how inadequate fixation can lead to the formation of artefactual clustering of receptors in lymphatic endothelial cells, focussing on the transmembrane hyaluronan receptors LYVE-1 and CD44, and the homotypic adhesion molecule CD31, each of which displays their native diffuse surface distribution pattern only when visualised with the right fixation techniques, i.e. PFA/GA in combination. Fluorescence recovery after photobleaching (FRAP) confirms that the artefactual receptor clusters are indeed introduced by residual mobility. In contrast, we observed full immobilisation of membrane proteins in cells that were fixed and then subsequently permeabilised, irrespective of whether the fixative was PFA or PFA/GA in combination. Our study underlines the importance of choosing appropriate sample preparation protocols for preserving authentic receptor organisation in advanced fluorescence microscopy.
authors.affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.
authors.forename: Tess A
authors.initials: TA
authors.lastname: Stanly
authors.affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.
authors.forename: Marco
authors.initials: M
authors.lastname: Fritzsche
authors.affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.
authors.forename: Suneale
authors.initials: S
authors.lastname: Banerji
authors.affiliation: Wolfson Imaging Centre, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.
authors.forename: Esther
authors.initials: E
authors.lastname: García
authors.affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK Science & Technology Facilities Council, Rutherford Appleton Laboratory, Central Laser Facility, Research Complex at Harwell, Harwell-Oxford Campus, Oxford OX11 0FA, UK.
authors.forename: Jorge
authors.initials: J
authors.lastname: Bernardino de la Serna
authors.affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK david.jackson@imm.ox.ac.uk christian.eggeling@rdm.ox.ac.uk.
authors.forename: David G
authors.initials: DG
authors.lastname: Jackson
authors.affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK Wolfson Imaging Centre, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK david.jackson@imm.ox.ac.uk christian.eggeling@rdm.ox.ac.uk.
authors.forename: Christian
authors.identifier: http://orcid.org/0000-0002-3698-5599
authors.initials: C
authors.lastname: Eggeling
keywords: FRAP; Fixation; Immunolabelling; LYVE-1; Membrane receptors; Receptor clustering; Super-resolution