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DOI: 10.1369/jhc.4A6605.2005
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Journal of Histochemistry and Cytochemistry
Volume 53 (7): 821-832, 2005
Copyright ©The Histochemical Society, Inc.

Light and Electron Microscopic Immunohistochemical Detection of Bromodeoxyuridine-labeled Cells in the Brain : Different Fixation and Processing Protocols

Laura B. Ngwenya, Alan Peters and Douglas L. Rosene

Department of Anatomy & Neurobiology, Boston University School of Medicine, Boston, Massachusetts (LBN,AP,DLR), and Yerkes National Primate Research Center, Emory University, Atlanta, Georgia (AP,DLR)

Correspondence to: Laura B. Ngwenya, Department of Anatomy & Neurobiology, Boston University School of Medicine, 715 Albany St., Boston, MA 02118. E-mail: ngwenya{at}bu.edu

Bromodeoxyuridine (BrdU) immunohistochemistry is the method of choice for labeling newly generated cells in the brain. Most BrdU studies utilize paraformaldehyde-fixed brain tissue because of its compatibility with both BrdU and other immunohistochemical methods. However, stronger fixation is required for electron microscopic studies, and unfixed tissue is needed for biochemical and molecular studies. Because there are no systematic studies comparing the effects of different fixatives on BrdU immunohistochemistry in brain tissue, we compared BrdU immunohistochemical methods in brain tissue fixed with 4% paraformaldehyde, a mixed glutaraldehyde–paraformaldehyde fixative for electron microscopy, and unfixed tissue from brains perfused only with buffer and flash frozen. After optimizing immunostaining protocols, qualitative assessments of light microscopic diaminobenzidine labeling and of double-label immunofluorescence with confocal microscopy demonstrated excellent BrdU labeling in each of the three groups. Quantitative stereological assessment of the number of BrdU-labeled cells in rat dentate gyrus showed no significant difference in the number of labeled cells detected with each perfusion protocol. Additionally, we developed a protocol to visualize BrdU-labeled cells in the electron microscope with adequate preservation of fine structure in both rat and monkey brain.

(J Histochem Cytochem 53:821–832, 2005)

Key Words: bromodeoxyuridine • ultrastructure • electron microscopy • glutaraldehyde • dentate gyrus • adult neurogenesis


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