Improved protocol for processing stented porcine coronary arteries for immunostaining.

摘要

Percutaneous coronary intervention has resulted in a paradigm shift in the treatment of coronary artery disease and myocardial infarction. However, neither bare-metal stents nor polymer-coated drug-eluting stents represent ideal therapies at this time due to the undesired in-stent stenosis or delayed thrombosis. Hence there is pressing clinical need for greater understanding of the cellular mechanisms involved. It is hoped that this in turn will provide insight into designing and developing the next generation of stents. Although immunohistochemistry and immunofluorescence are appropriate tools in understanding the molecular histology, performing these techniques on stented blood vessels is technically challenging because of poor permeability of antibodies into the stented blood vessels which are embedded in methacrylate-based resins and inadequate image resolution due to autofluorescence. Hence there is a need to develop techniques which can facilitate immunohistochemistry/immunofluorescence procedures on stented blood vessel cross-sections. In this study we describe an improved protocol for processing stented porcine coronary arteries for immunostaining with smooth muscle cell, endothelial cell, monocyte and macrophage markers. We first identified the optimal conditions for resin embedding of stented artery and cross sectioned the vessels using high speed precision wafering diamond blade. The sections were then ground using two levels of water sandpaper on a Metaserve 2000 grinder to achieve the desired thickness. For immunostaining, we developed a novel deplasticization protocol which favors optimal antibody permeabilization. Our protocol not only provides feasibility of improved immunostaining of stented artery sections but also results in high quality images.