Studies of childhood brain tumors using immunohistochemistry and microwave technology: methodological considerations

The immunohistochemical detection of antigens in archival tissue sections has been hampered by the poor reactivity of certain polypeptides in conventional formalin-fixed, paraffin-embedded material. For example, the poor reactivity of neurofilament proteins (NFPs) in surgical and autopsy specimens has been a major drawback of previous large, retrospective, clinicopathologic studies of pediatric primitive neuroectodermal tumors (PNETs), also known as medulloblastomas. We report our experience with a method of antigen retrieval which greatly enhanced the immunohistochemical detection of neuronal and glial intermediate filament proteins, retinal S-antigen (RSA), and the proliferating cell nuclear antigen (PCNA) in archival, paraffin-embedded, formalin or Bouin's-fixed, pediatric brain tumors, particularly PNETs. The technique involves adding a single brief step to an established avidin-biotin complex (ABC) immunohistochemical protocol (Vectastain Elite Kit). This step involves boiling tissue sections in distilled water for 5 min in a microwave oven. The specificity of staining was consistent with known cell and tissue specificities of the well-characterized monoclonal antibodies used and there was minimal background. Synaptophysin (SYN) staining was unaffected by heating and immunoreactivity of the low affinity nerve growth factor (p75NGFR) and the neural cell adhesion molecule (NCAM) were lost. The enhanced detection of neuronal and glial antigens in routinely prepared, formalin-fixed, archival material should facilitate large retrospective clinicopathologic studies designed to assess the prognostic implications of differentiation in PNETs and to better understand the biology of these tumors.