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Production and Histological Application of Affinity-purified Antibodies to Heat-denatured Green Fluorescent Protein

Kouichi C. Nakamura, Hiroshi Kameda, Yoshinori Koshimizu, Yuchio Yanagawa and Takeshi Kaneko

Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Kyoto, Japan (KCN,HK,YK,TK); Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Kawaguchi, Japan (KCN,TK); and Department of Genetic and Behavioral Neuroscience, Gunma University Graduate School of Medicine, Maebashi, Japan (YY)

Correspondence to: Takeshi Kaneko, Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan. E-mail: kaneko{at}mbs.med.kyoto-u.ac.jp

Enhanced green fluorescent protein (GFP) irreversibly loses not only fluorescence but also antigenicity recognized with conventional anti-GFP antibodies by heat denaturation. This hinders combinatory applications of the GFP immunodetection technique with heat-requiring procedures, such as in situ hybridization histochemistry, antigen retrieval, and Western blot. Here we produced new rabbit and guinea pig antibodies against heat-denatured GFP. The polyclonal antibodies affinity-purified with the antigen column detected a single band corresponding to the molecular size of GFP in Western blot analysis, with mouse brain expressing GFP from the GAD67 locus. By immunofluorescence labeling, the new antibodies detected GFP molecules in heat (70C)-treated sections but not in untreated sections of the mouse brain. When the sections were incubated at 37C with in situ hybridization buffer containing 50% formamide, a denaturing reagent, the sections lost immunoreactivity with the conventional anti-GFP antibodies but acquired immunoreactivity with the new antibodies to heat-denatured GFP. Finally, GFP immunofluorescence was successfully visualized with the new antibodies in sections of the GFP-expressing mice labeled by fluorescence in situ hybridization histochemistry against GAD67 mRNA. Thus, the antibodies produced in this study may provide an opportunity to combine GFP immunodetection with procedures requiring heat treatment. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 56:647–657, 2008)

Key Words: immunofluorescence • in situ hybridization • fluorescence microscopy • antigen retrieval • Western blot

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