title: Production and histological application of affinity-purified antibodies to heat-denatured green fluorescent protein.
abstract: 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 (> or = 70 degrees C)-treated sections but not in untreated sections of the mouse brain. When the sections were incubated at > or = 37 degrees C 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.
authors.affiliation: Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
authors.forename: Kouichi C
authors.initials: KC
authors.lastname: Nakamura
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kameda
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Koshimizu
authors.forename: Yuchio
authors.initials: Y
authors.lastname: Yanagawa
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kaneko
mesh_terms: D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D001921:Brain / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D005968:Glutamate Decarboxylase / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006168:Guinea Pigs; D006358:Hot Temperature; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D009474:Neurons / Q000378:metabolism; D011489:Protein Denaturation; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits
chemical_list: D000906:Antibodies; D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1