Stanly Tess A, Fritzsche Marco, Banerji Suneale, García Esther, Bernardino de la Serna Jorge, Jackson David G, Eggeling Christian
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.
Biol Open. 2016 Sep 15;5(9):1343-50. doi: 10.1242/bio.019943.
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.
已知受体聚集会触发信号转导事件，这些事件会导致细胞功能发生关键变化。通过荧光显微镜对这类聚集体进行准确成像，依赖于在免疫标记之前应用适当的细胞固定方法，以避免抗体自身造成人为的重新分布。先前的研究强调，单独使用多聚甲醛（PFA）固定不足以有效固定膜相关分子，而PFA与戊二醛（GA）联合固定具有优势。在此，我们利用荧光显微镜突出显示了固定不足如何导致淋巴管内皮细胞中受体形成人为聚集，重点关注跨膜透明质酸受体LYVE-1和CD44，以及同型粘附分子CD31，只有在使用正确的固定技术（即PFA/GA联合）进行可视化时，它们各自才会呈现天然的弥散性表面分布模式。光漂白后荧光恢复（FRAP）证实，人为的受体聚集体确实是由残留的流动性引入的。相比之下，我们观察到，无论固定剂是PFA还是PFA/GA联合，在固定然后随后进行通透处理的细胞中，膜蛋白都能完全固定。我们的研究强调了选择合适的样品制备方案对于在先进的荧光显微镜下保留真实受体组织的重要性。
