Twamley Shailey Gale, Stach Anke, Heilmann Heike, Söhl-Kielczynski Berit, Stangl Verena, Ludwig Antje, Münster-Wandowski Agnieszka
Medizinische Klinik für Kardiologie und Angiologie, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 10117 Berlin, Germany.
DZHK (German Centre for Cardiovascular Research), Partner Site, 10117 Berlin, Germany.
Biology (Basel). 2021 May 4;10(5):402. doi: 10.3390/biology10050402.
The glycocalyx (GCX), a pericellular carbohydrate rich hydrogel, forms a selective barrier that shields the cellular membrane, provides mechanical support, and regulates the transport and diffusion of molecules. The GCX is a fragile structure, making it difficult to study by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Sample preparation by conventional chemical fixation destroys the GCX, giving a false impression of its organization. An additional challenge is to process the GCX in a way that preserves its morphology and enhanced antigenicity to study its cell-specific composition. The aim of this study was to provide a protocol to preserve both antigen accessibility and the unique morphology of the GCX. We established a combined high pressure freezing (HPF), osmium-free freeze substitution (FS), rehydration, and pre-embedding immunogold labeling method for TEM. Our results showed specific immunogold labeling of GCX components expressed in human monocytic THP-1 cells, hyaluronic acid receptor (CD44) and chondroitin sulfate (CS), and maintained a well-preserved GCX morphology. We adapted the protocol for antigen localization by CLSM and confirmed the specific distribution pattern of GCX components. The presented combination of HPF, FS, rehydration, and immunolabeling for both TEM and CLSM offers the possibility for analyzing the morphology and composition of the unique GCX structure.
糖萼（GCX）是一种富含碳水化合物的细胞周围水凝胶，形成了一个选择性屏障，可保护细胞膜、提供机械支持并调节分子的运输和扩散。糖萼是一种脆弱的结构，使得通过透射电子显微镜（TEM）和共聚焦激光扫描显微镜（CLSM）进行研究变得困难。传统化学固定法制备样品会破坏糖萼，从而对其结构产生错误的认识。另一个挑战是要以一种既能保留其形态又能增强其抗原性的方式来处理糖萼，以便研究其细胞特异性组成。本研究的目的是提供一种既能保留抗原可及性又能保持糖萼独特形态的方法。我们建立了一种用于透射电子显微镜的联合高压冷冻（HPF）、无锇冷冻置换（FS）、复水和预包埋免疫金标记方法。我们的结果显示，在人单核细胞THP-1细胞中表达的糖萼成分、透明质酸受体（CD44）和硫酸软骨素（CS）有特异性免疫金标记，并且保持了保存良好的糖萼形态。我们将该方法应用于通过共聚焦激光扫描显微镜进行抗原定位，并证实了糖萼成分的特异性分布模式。所提出的用于透射电子显微镜和共聚焦激光扫描显微镜的高压冷冻、冷冻置换、复水和免疫标记的组合方法，为分析独特的糖萼结构的形态和组成提供了可能性。
