Development of a Protocol for Triple Labeling of MAP-2, Fluoro-Jade, and DAPI in a Single Paraffin-Embedded Rat Brain Section
Technical rept. Jan-Jun 2004
ARMY MEDICAL RESEARCH INST OF CHEMICAL DEFENSE ABERDEEN PROVING GROUND MD
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The present study was conducted to develop a protocol for triple labeling of MAP-2, Fluoro-Jade, and DAPI to evaluate the pathological consequences of nerve agent toxicity in the rat model. Brains of rats subcutaneously challenged with a single injection of soman were formalin-fixed, paraffin-embedded, and sectioned. Following microwave antigen retrieval, sections were stained with MAP-2 monoclonal antibody to evaluate changes in dendritic processes, with Fluoro-Jade to examine the degree of neuronal cell degeneration or death, and with DAPI to assess the number of viable nuclei. Control hippocampus showed typical MAP-2 immunofluorescence localized predominantly in dendritic processes, numerous DAPI positive nuclei, and absence of Fluoro-Jade positive neurons. In the hippocampus of soman-exposed rats, Fluoro-Jade positive cells were detected in areas where MAP-2 immunofluorescence was lost and the number of DAPI positive nuclei was decreased. The results suggest that our triple labeling technique is effective for simultaneously examining MAP-2, Fluoro-Jade, and DAPI fluorescence in the same section, thus reducing the number of sections necessary to comprehensively evaluate dendritic pathology, neurodegeneration, and neuronal loss. More importantly, the technique permits precise anatomical merging of MAP-2, Fluoro-Jade, and DAPI images, which cannot be accomplished when these stains are performed separately on serial sections.
- Anatomy and Physiology
- Medicine and Medical Research