Automated procedure for in situ detection of apoptosis

Apoptosis, or programmed cell death, occurs during embryonic development, normal tissue homeostasis, and oncogenesis and as a result of toxic insult. Understanding the role that apoptosis plays is important in learning more about the mechanisms of these normal and abnormal processes. Several assays have been developed to label cells undergoing apoptosis, but the assays are labor intensive and sometimes lack in reproducibility. We describe a 76 step automated procedure that labels apoptotic cells in 4 mu m, formalin fixed tissues. The automated procedure uses capillary action and a commercially available peroxidase-based kit to do enzymatic nucleotidyl end-labeling of DNA fragments and immunohistochemical 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. Approximately 85-95% of cells or cellular fragments that were morphologically consistent with apoptotic cells or apoptotic bodies stained golden-brown immunohistochemically with diaminobenzidine. Automation of in situ detection of apoptosis allows processing of 20-40 slides in a single run, reducing assay variability, conserve the technologist's time and effort, and reduce the quantity of reagents required.