Abstract
Glycocalyx (GCX) is a thin layer of negatively charged glycoproteins that covers the vascular endothelial surface and regulates various biological processes. Because of the delicate and fragile properties of this structure, it is difficult to detect GCX morphologically. We established a simple method for a three-dimensional visualization of endothelial GCX using low-vacuum scanning electron microscopy (LVSEM) on formalin-fixed paraffin-embedded (FFPE) sections. Mouse kidney tissue was fixed with 10% buffered formalin containing 1% Alcian blue (ALB) via perfusion and immersion. FFPE sections were observed by light microscopy (LM) and LVSEM, and formalin-fixed epoxy resin-embedded ultrathin sections were observed by transmission electron microscopy (TEM). The endothelial GCX from various levels of kidney blood vessels was stained blue in LM and confirmed as a thin osmiophilic layer in TEM. In LVSEM, the sections stained by periodic acid methenamine silver (PAM) revealed the endothelial GCX as a layer of dense silver-enhanced particles, in both the samples fixed via perfusion and immersion. Correlative light and electron microscopy (CLEM) revealed the fine visible structure of endothelial GCX. This simple method using FFPE samples with ALB will enable the three-dimensional evaluation of endothelial GCX alterations in various human diseases associated with endothelial injury in future studies.
Highlights
Endothelial glycocalyx (GCX) is a thin layer of glycoproteins covering the luminal surface of vascular endothelial cells and is associated with various important biological functions, including cell adhesion, migration, differentiation, and inflammation [1,2,3,4]
Several clinical biomarkers for GCX injury have been reported, such as serum concentration of syndecan-1, heparin sulfate, and hyaluronic acid, reflecting the degrees of their shedding off from the endothelial surface into the bloodstream [22,23,24]. These markers may be useful for the evaluation of systemic injury of endothelial GCX, but evaluation of local injury in each organ and tissue is necessary for the evaluation of pathogenesis of organspecific diseases associated with endothelial injury
The advantages of this technique for Electron microscopy (EM) are that it requires less time for EM specimen preparation, provides greater convenience for the use of the same sample for light microscopy (LM), allows a wide range of observations using a large specimen for LM, allows threedimensional observation and it can be used on previously obtained pathological samples (FFPE blocks)
Summary
Endothelial glycocalyx (GCX) is a thin layer of glycoproteins covering the luminal surface of vascular endothelial cells and is associated with various important biological functions, including cell adhesion, migration, differentiation, and inflammation [1,2,3,4]. Because of its fragile properties, visualization of endothelial GCX is difficult using traditional light and electron microscopic procedures. To combat this issue, several techniques using cationic molecules that bind. Medical Molecular Morphology (2021) 54:95–107 investigation of alterations in the endothelial glycocalyx in various human diseases associated with endothelial injury
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