AUTOMATED PROCEDURE FOR IN-SITU DETECTION OF APOPTOSIS

Apoptosis, or programmed cell death, occurs during embryonic developme nt, normal tissue homeostasis, and oncogenesis and as a result of toxi c insult. Understanding the role that apoptosis plays is important in learning more about the mechanisms of these normal and abnormal proces ses. Several assays have been developed to label cells undergoing apop tosis, but the assays are labor intensive and sometimes lack in reprod ucibility. We describe a 76 step automated procedure that labels apopt otic cells in 4 mu m, formalin fixed tissues. The automated procedure uses capillary action and a commercially available peroxidase-based ki t to do enzymatic nucleotidyl end-labeling of DNA fragments and immuno histochemical detection of apoptosis. Apoptotic cells were labeled in 4 different tissue types (liver, mammary gland, uterus, and limb bud), illustrating the general applicability of the automated procedure. Ap proximately 85-95% of cells or cellular fragments that were morphologi cally consistent with apoptotic cells or apoptotic bodies stained gold en-brown immunohistochemically with diaminobenzidine. Automation of in situ detection of apoptosis allows processing of 20-40 slides in a si ngle run, reducing assay variability, conserve the technologist's time and effort, and reduce the quantity of reagents required.