研究目的
To develop a new method for combined observation of serial sections of stented arteries embedded in resin by light microscopy and transmission electron microscopy to relate histopathological changes at different scales.
研究成果
The developed method successfully enables combined observation of stented arteries by LM and TEM, allowing correlation of histopathological changes from millimeter to nanometer scales. It provides detailed insights into endothelial recovery and could be useful for evaluating biocompatibility of medical devices, suggesting future applications in stent development and other implants.
研究不足
The method may have technical difficulties with tissues including hard materials, and the processing is complex and time-consuming. Potential areas for optimization include improving staining quality and applicability to other medical devices.
1：Experimental Design and Method Selection:
The study designed a method using Quetol 651 resin embedding and periodic acid/hydrogen peroxide for osmium removal to enable combined LM and TEM observation of stented arteries.
2：Sample Selection and Data Sources:
Porcine coronary arteries with implanted stents were used, approved by the Institutional Animal Care and Use Committee.
3：List of Experimental Equipment and Materials:
Equipment includes a diamond band saw (V-19; Luxo), rotary microtome (RM2255; Leica), ultramicrotome with diamond knife (Diatome), TEM (HT7700; Hitachi High-Technologies), slide press (object holder press; Kulzer), and materials such as Quetol 651 resin, Quetol 812 resin, fixatives (paraformaldehyde, glutaraldehyde, osmium tetroxide), stains (hematoxylin and eosin, Elastica–Masson trichrome), and chemicals (periodic acid, hydrogen peroxide).
4：Experimental Procedures and Operational Workflow:
Hearts were perfusion fixed, stented arteries removed and fixed, embedded in resin, sectioned serially, deplasticized, treated for osmium removal, stained for LM, and re-embedded and sectioned for TEM using the resupinate method.
5：Data Analysis Methods:
Observations were made using LM and TEM to assess histopathological features and ultrastructure.
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rotary microtome
RM2255
Leica
Used for sectioning resin-embedded tissues into serial sections for microscopy.
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transmission electron microscope
HT7700
Hitachi High-Technologies
Used to observe ultrastructural features of tissue sections.
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Quetol 651
651
Nisshin EM
Embedding medium for tissue sections, used for both light microscopy and transmission electron microscopy due to its low viscosity and good sectioning properties.
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Quetol 812
812
Nisshin EM
Resin used for re-embedding thin sections for transmission electron microscopy.
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diamond band saw
V-19
Luxo
Used to cut polymerized resin blocks containing stented arteries.
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ultramicrotome
Used with a diamond knife to cut ultrathin sections for transmission electron microscopy.
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diamond knife
Diatome
Used on the ultramicrotome to cut thin sections for TEM.
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slide press
object holder press
Kulzer
Used to press and flatten sections on glass slides for light microscopy.
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polypropylene tube
PP-10
Maruemu
Used for embedding tissues in resin during polymerization.
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