Christina Mayerl, Angelika Hammerer-Lercher, Bernd Puschendorf, Martina Prelog
Division of Experimental Pathophysiology and Immunology, Biocenter Innsbruck, Innsbruck Medical University, Austria.
J Immunol Methods. 2006 Oct 20;316(1-2):163-6. doi: 10.1016/j.jim.2006.08.013. Epub 2006 Sep 20.
The TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) technique has been described as a sensitive method for detection of apoptotic nuclei in tissues and preferential staining of apoptotic strand breaks. Short-term microwave pre-treatment, a non-enzymatic pre-treatment technique of antigen retrieval, has been demonstrated to optimize the TUNEL method for in situ detection of apoptotic cells in formalin-fixed paraffin-embedded tissue sections. In the present study, we sensitized internal mammary artery sections by short-term microwave pre-treatment and used a two-step indirect enzymatic method to gain as an end product differentially stained cells, namely TUNEL-positive cells and these positive for the surface marker von Willebrand factor (vWF). This technique enables to clearly distinguish between apoptotic, non-apoptotic and vWF-positive cells that are phenotypic for endothelial cells. Phenotypic identification of cells is simplified by double staining with cell surface markers. This rapid, sensitive and reproducible technique allows simultaneous detection of DNA fragmentation and phenotypic markers in the same paraffin-embedded human tissue section.
TUNEL（末端脱氧核苷酸转移酶介导的dUTP缺口末端标记）技术已被描述为一种检测组织中凋亡细胞核以及优先染色凋亡链断裂的灵敏方法。短期微波预处理作为一种非酶促抗原修复预处理技术，已被证明可优化TUNEL方法，用于福尔马林固定石蜡包埋组织切片中凋亡细胞的原位检测。在本研究中，我们通过短期微波预处理使乳内动脉切片致敏，并采用两步间接酶法，以获得差异染色细胞作为终产物，即TUNEL阳性细胞和表面标志物血管性血友病因子（vWF）阳性细胞。该技术能够清晰区分凋亡细胞、非凋亡细胞以及具有内皮细胞表型的vWF阳性细胞。通过与细胞表面标志物进行双重染色可简化细胞的表型鉴定。这种快速、灵敏且可重复的技术能够在同一石蜡包埋的人体组织切片中同时检测DNA片段化和表型标志物。
