Simultaneous detection of a cell surface antigen and apoptosis by microwave-sensitized TUNEL assay on paraffin sections

Abstract

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.