Detection of fragmented DNA in apoptotic cells embedded in LR White: A combined histochemical (LM) and ultrastructural (EM) study

We developed an improved method for the detection of double-strand DNA brea ks in apoptotic cells at both the light (LM) and electron microscopic (EM) levels using a modification of the TdT (terminal deoxynucleotidyl transfera se)-mediated dUTP nick end-labeling (TUNEL) technique. Cultured rat cerebel lar granule cells were exposed to low potassium conditions to induce apopto sis. Twenty-four hr after treatment, one group of cells was fixed in situ w ith 4% paraformaldehyde and labeled for DNA fragmentation characteristic of apoptosis. Apoptotic cells were visualized with diaminobenzidine (DAB) and viewed by LM. The second group of cells was detached from the culture dish , pelleted, fixed with a 4% paraformaldehyde and 0.2% glutaraldehyde mixtur e, and embedded in LR White. For LM, the modified TUNEL technique was perfo rmed on 1.5-mu m LR White sections and apoptotic cells were visualized usin g an enzymatic reaction to generate a blue precipitate. For EM, thin sectio ns (94 nm) were processed and DNA fragmentation was identified using modifi ed TUNEL with streptavidin-conjugated gold in conjunction with in-depth ult rastructural detail. Alternate sections of cells embedded in LR White can t herefore be used for LM and EM TUNEL-based detection of apoptosis. The pres ent findings suggest that the modified TUNEL technique on LR White semithin and consecutive thin sections has useful application for studying the fund amental mechanism of cell death.