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