Pre-embedding immunogold labeling of TUNEL stain enables evaluation of DNA strand breaks and ultrastructural alterations in individual cells of neuronal tissue

Abstract

In the brain apoptosis may occur as a physiological phenomenon during periods of programmed cell death as well as under pathological conditions such as ischemia, trauma, tumor, and degenerative diseases. While the definition of apoptotic cell death was originally based on ultrastructural alterations, the detection of DNA double-strand breaks has become an important feature in studies of apoptosis. Currently, the terminal transferase-mediated dUTP nick-end labeling (TUNEL) procedure is widely used for detection of apoptotic cell death. However, there is a growing body of evidence to suggest that the TUNEL staining does not label apoptotic alterations exclusively. Therefore, a new staining procedure was developed combining TUNEL methodology with pre-embedding nanogold labeling to detect DNA double-strand breaks in individual cells by electron microscopy and assess the accompanying ultrastructural alterations. In vitro DNAse-treated vibratome sections (thickness, 20 micro m) from normal adult rat brains were used to develop the staining procedure consisting of the following steps: (i) TUNEL staining of free-floating vibratome sections using fluorescein isothiocyanate (FITC)-labeled UTP, (ii) conversion of the fluorescence signal into an electron-dense signal using an anti-FITC antibody coupled with ultrasmall (diameter, 0.8 nm) gold particles followed by silver enhancement, and (iii) osmification, embedding in Spurr resin and cutting of ultrathin sections. Early postnatal brain tissue was used to study physiologically occurring apoptotic cell death. Under these conditions different patterns of gold staining were observed probably representing different states of cellular decay along the apoptotic avenue. Severe focal brain ischemia was studied as a pathological situation in which intense TUNEL staining occurs. Under these conditions TUNEL labeling of cells was regularly observed in conjunction with ultrastructural alterations indicative of necrosis. These results suggest that under pathological conditions apoptosis and necrosis are not mutually exclusive mechanisms but rather may occur concurrently along a continuum in which cell death occurs.