The use of proliferating cell nuclear antigen immunohistochemistry with a unique functional marker to detect postnatal neurogenesis in paraffin-embedded sections of the mature pig brain

Abstract

Until recently, evidence supporting postnatal neurogenesis was controversial. Much of the debate has centered on the identification of the dividing cells as neurons versus glia. Because neurogenesis has become a well-documented phenomenon, there is a need for reliable protocols to identify recently divided neurons in a wide range of situations. To facilitate the investigation of postnatal neurogenesis of magnocellular neurons in the pig hypothalamus, a sequential immunohistochemical staining technique was developed for use on serial sections of paraffin-embedded tissue. Proliferating neurons were labeled using mouse-derived monoclonal antibodies to detect proliferating cell nuclear antigen (PCNA) and vasopressin (VP). PCNA, a nuclear protein essential for cell division, identifies recently divided cells in the brains of healthy animals. VP is a unique functional marker for a mature neuron. The presence of a cell with VP positive cytoplasm and a PCNA positive nucleus demonstrates the presence of a VP-producing neuron that has recently divided. This protocol allowed us to safely and accurately label recently proliferated neurons in the mature pig hypothalamus and can be used on archived tissue. This data can be used for further morphometric analysis, as serial sectioning allows for three-dimensional reconstruction of hypothalamic nuclei.