Abstract
Currently, an ultrastructural analysis of cardiovascular tissues is significantly complicated. Routine histopathological examinations and immunohistochemical staining suffer from a relatively low resolution of light microscopy, whereas the fluorescence imaging of plaques and bioprosthetic heart valves yields considerable background noise from the convoluted extracellular matrix that often results in a low signal-to-noise ratio. Besides, the sectioning of calcified or stent-expanded blood vessels or mineralised heart valves leads to a critical loss of their integrity, demanding other methods to be developed. Here, we designed a conceptually novel approach that combines conventional formalin fixation, sequential incubation in heavy metal solutions (osmium tetroxide, uranyl acetate or lanthanides, and lead citrate), and the embedding of the whole specimen into epoxy resin to retain its integrity while accessing the region of interest by grinding and polishing. Upon carbon sputtering, the sample is visualised by means of backscattered scanning electron microscopy. The technique fully preserves calcified and stent-expanded tissues, permits a detailed analysis of vascular and valvular composition and architecture, enables discrimination between multiple cell types (including endothelial cells, vascular smooth muscle cells, fibroblasts, adipocytes, mast cells, foam cells, foreign-body giant cells, canonical macrophages, neutrophils, and lymphocytes) and microvascular identities (arterioles, venules, and capillaries), and gives a technical possibility for quantitating the number, area, and density of the blood vessels. Hence, we suggest that our approach is capable of providing a pathophysiological insight into cardiovascular disease development. The protocol does not require specific expertise and can be employed in virtually any laboratory that has a scanning electron microscope.
Highlights
In spite of the variety of approaches for ultrastructural pathology, which generally include the preparation of formalinfixed paraffin-embedded, snap-frozen, or fresh tissue specimens further stained with specific dyes or chromogen, fluorescent, or gold-labelled antibodies and visualised by light, epifluorescence, confocal, or electron microscopy [1,2,3], the processing and imaging of calcified or stent-expanded cardiovascular tissue remain poor [4]
We developed and validated a conceptually novel histological approach that couples whole-specimen formalin fixation with heavy metal staining, epoxy resin embedding, grinding, and polishing of epoxy resin blocks, and backscattered scanning electron microscopy (BSEM). As this technique fully retains the integrity of calcified or stentexpanded tissues and combines high-magnification visualisation, rapid image acquisition, and the possibility to perform elemental analysis, we suggest it as an optimal solution for cardiovascular research, especially for studies on calcification and microcirculation
Embedding into Epoxy Resin and Preparation of the Sample for BSEM (Steps 19–24) Here, we describe a critical step of further sample retrieval by grinding and polishing and the final preparations that ensure high-quality BSEM
Summary
In spite of the variety of approaches for ultrastructural pathology, which generally include the preparation of formalinfixed paraffin-embedded, snap-frozen, or fresh tissue specimens further stained with specific dyes or chromogen, fluorescent, or gold-labelled antibodies and visualised by light, epifluorescence, confocal, or electron microscopy [1,2,3], the processing and imaging of calcified or stent-expanded cardiovascular tissue remain poor [4]. We developed and validated a conceptually novel histological approach that couples whole-specimen formalin fixation with heavy metal staining (i.e., incubation in osmium tetroxide, uranyl acetate, and lead citrate solutions), epoxy resin embedding, grinding, and polishing of epoxy resin blocks, and backscattered scanning electron microscopy (BSEM). As this technique fully retains the integrity of calcified or stentexpanded tissues and combines high-magnification visualisation, rapid image acquisition, and the possibility to perform elemental analysis, we suggest it as an optimal solution for cardiovascular research, especially for studies on calcification and microcirculation. Keep away from heat/sparks/open flames/hot surfaces, do not smoke around reagent, use explosionproof electrical/ventilating/lighting/equipment, use only nonsparking tools, take precautionary measures against static discharge, wear protective clothing (gloves and face protection), wash gloves and hands thoroughly after handling, and use only in a well-ventilated area such as a fume hood
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