Boulanger Jenna J, Staines William A, LeBlanc Véronique, Khoo Eve-Ling, Liang Jacky, Messier Claude
School of Psychology, University of Ottawa, 136 Jean-Jacques Lussier, Ottawa, ON, Canada K1 N 6N5.
Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada.
J Neurosci Methods. 2016 Feb 1;259:40-46. doi: 10.1016/j.jneumeth.2015.11.006. Epub 2015 Nov 24.
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.
用于揭示5-溴-2'-脱氧尿苷（BrdU）掺入的典型免疫组织化学技术需要通过加热和酸使DNA变性以通透细胞核。这种处理会损害组织并降低几种蛋白质的抗原性，进而导致免疫染色减弱和/或假阴性。我们发现，在针对感兴趣的抗原进行免疫组织化学后进行过夜固定步骤，可在随后用于BrdU染色的酸/热变性处理过程中保护免疫染色。我们使用该技术研究了NG2CreER:EYFP报告基因小鼠中最近分裂的少突胶质细胞祖细胞的分化。我们使用GFP抗EYFP抗体以最大程度地观察含EYFP的少突胶质细胞祖细胞、少突胶质细胞转录因子1（Olig1），并使用谷胱甘肽S-转移酶π（GST-pi）来确认细胞表型。DNA变性会使GFP、Olig1和GST-pi的免疫染色减弱。我们发现，在DNA变性之前，对GFP/Olig1和GFP/GST-pi进行双重免疫染色后加入固定步骤，可防止与此处理相关的褪色和假阴性。在BrdU免疫组织化学方案中简单增加这一步骤，扩展了可与BrdU联合检测的蛋白质范围，以及可在细胞增殖研究中成功使用的抗体数量。
