title: A simple histological technique to improve immunostaining when using DNA denaturation for BrdU labelling.
abstract: The typical immunohistochemistry technique used to reveal 5-bromo-2'-deoxyuridine (BrdU) incorporation requires denaturation of the DNA by heat and acid to permeabilize the cell nucleus. This treatment can damage tissue and reduce the antigenicity of several proteins, which then leads to weak immunostaining and/or false negatives. We show that an overnight post-fixation step following immunohistochemistry for antigens of interest protects immunostaining during the acid/heat denaturation treatment for subsequent BrdU staining. We used this technique to study the differentiation of recently divided oligodendrocyte progenitor cells in NG2CreER:EYFP reporter mice. We used a GFP anti-EYFP antibody to maximize visualization of the EYFP-containing oligodendrocyte progenitor cells, Olig1, and GST-pi to confirm the cell phenotype. Immunostaining for GFP, Olig1, and GST-pi is reduced by DNA denaturation. We found that incorporating a post-fixation step after double immunostaining for GFP/Olig1 and GFP/GST-pi prior to DNA denaturation prevented the fading and false negatives associated with this treatment. This simple addition to BrdU immunohistochemistry protocols extends the range of proteins that can be detected in combination with BrdU, along with the number of antibodies that can be used successfully in the study of cell proliferation.
authors.affiliation: School of Psychology, University of Ottawa, 136 Jean-Jacques Lussier, Ottawa, ON, Canada K1 N 6N5.
authors.forename: Jenna J
authors.initials: JJ
authors.lastname: Boulanger
authors.affiliation: Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada.
authors.forename: William A
authors.initials: WA
authors.lastname: Staines
authors.affiliation: School of Psychology, University of Ottawa, 136 Jean-Jacques Lussier, Ottawa, ON, Canada K1 N 6N5.
authors.forename: Véronique
authors.initials: V
authors.lastname: LeBlanc
authors.affiliation: School of Psychology, University of Ottawa, 136 Jean-Jacques Lussier, Ottawa, ON, Canada K1 N 6N5.
authors.forename: Eve-Ling
authors.initials: EL
authors.lastname: Khoo
authors.affiliation: School of Psychology, University of Ottawa, 136 Jean-Jacques Lussier, Ottawa, ON, Canada K1 N 6N5.
authors.forename: Jacky
authors.initials: J
authors.lastname: Liang
authors.affiliation: School of Psychology, University of Ottawa, 136 Jean-Jacques Lussier, Ottawa, ON, Canada K1 N 6N5. Electronic address: cmessier@uottwa.ca.
authors.forename: Claude
authors.initials: C
authors.lastname: Messier
mesh_terms: D000818:Animals; D000963:Antimetabolites; D001973:Bromodeoxyuridine; D004247:DNA / Q000378:metabolism*; D007150:Immunohistochemistry / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D058953:Neural Stem Cells / Q000378:metabolism*; D055495:Neurogenesis; D009691:Nucleic Acid Denaturation; D009836:Oligodendroglia / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*; D013936:Thymidine / Q000032:analysis*
chemical_list: D000963:Antimetabolites; D004247:DNA; D001973:Bromodeoxyuridine; D013936:Thymidine
keywords: BrdU; Denaturation; Gliogenesis; Immunohistochemistry; Neurogenesis