Journal
BIOLOGY OPEN
Volume 5, Issue 9, Pages 1343-1350Publisher
COMPANY BIOLOGISTS LTD
DOI: 10.1242/bio.019943
Keywords
Fixation; Immunolabelling; Membrane receptors; Receptor clustering; LYVE-1; FRAP; Super-resolution
Categories
Funding
- Wellcome Trust [104924/14/Z/14]
- Medical Research Council [G0902418, MC_UU_12025, MC_UU_12010/unit]
- Medical Research Council/Biotechnology and Biological Sciences Research Council/Engineering and Physical Sciences Research Council [MR/K01577X/1]
- Biotechnology and Biological Sciences Research Council , Deutsche Forschungsgemeinschaft-PerTrans consortium [BB/L00433X/1]
- European Union Seventh Framework Programme Marie Curie Career Integration Grant
- University of Oxford
- Biotechnology and Biological Sciences Research Council [BB/L00433X/1] Funding Source: researchfish
- Medical Research Council [MC_UU_12010/2, MC_U137884182, MR/K01577X/1, MC_UU_12010/9, 1093777, G0902418] Funding Source: researchfish
- Wellcome Trust [107457/Z/15/Z] Funding Source: researchfish
- BBSRC [BB/L014327/1] Funding Source: UKRI
- MRC [MC_UU_12010/9, G0902418, MC_UU_12010/2, MR/K01577X/1, MC_U137884182] Funding Source: UKRI
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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.
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