Search for conditions to detect epigenetic marks and nuclear proteins in immunostaining of the testis and cartilage
Search for conditions to detect epigenetic marks and nuclear proteins in immunostaining of the testis and cartilage
复制标题
寻找在睾丸和软骨免疫染色中检测表观遗传标记和核蛋白的条件
DOI：
10.1155/2014/658293
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发表时间：
2014
期刊：
影响因子：
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通讯作者：
Yoshiki Nakamura,et al
中科院分区：
文献类型：
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作者：
Hisashi Ideno;Akemi Shimada;Taichi Kamiunten;Kazuhiko Imaizumi;Yoshiki Nakamura,et al
核蛋白和修饰的组蛋白尾部的定位在组织和细胞水平的细胞分化期间发生变化。石蜡切片中的免疫染色是评价蛋白定位的最有效方法。由于核蛋白对固定敏感，因此应根据所用的特定抗体和组织优化免疫组织化学条件。在这项研究中，我们寻找最佳的条件来检测组蛋白修饰在组蛋白H3赖氨酸9（H3K9）和H3K9甲基转移酶G9a在睾丸和软骨的石蜡切片。在睾丸中，抗原修复（AR）是检测H3K9me1和me3、G9a和核蛋白增殖细胞核抗原（PCNA）所必需的。使用AR，固定时间越短，G9a和PCNA的检测结果越好。在没有AR的情况下，H3K9me2和H3K9ac可以在睾丸初级精母细胞中以较短的固定时间检测到。与睾丸相反，所有测试的抗体都可以检测到它们的表位，而不管生长板软骨中的AR应用。因此，表观遗传标记和核蛋白的检测条件应考虑固定时间和AR在不同组织和抗体中的应用进行优化。
The localization of nuclear proteins and modified histone tails changes during cell differentiation at the tissue as well as at the cellular level. Immunostaining in paraffin sections is the most powerful approach available to evaluate protein localization. Since nuclear proteins are sensitive to fixation, immunohistochemical conditions should be optimized in light of the particular antibodies and tissues employed. In this study, we searched for optimal conditions to detect histone modification at histone H3 lysine 9 (H3K9) and H3K9 methyltransferase G9a in the testis and cartilage in paraffin sections. In the testis, antigen retrieval (AR) was indispensable for detecting H3K9me1 and me3, G9a, and nuclear protein proliferating cell nuclear antigen (PCNA). With AR, shorter fixation times yielded better results for the detection of G9a and PCNA. Without AR, H3K9me2 and H3K9ac could be detected at shorter fixation times in primary spermatocytes of the testis. In contrast to the testis, all antibodies tested could detect their epitopes irrespective of AR application in the growth plate cartilage. Thus, conditions for the detection of epigenetic marks and nuclear proteins should be optimized in consideration of fixation time and AR application in different tissues and antibodies.