Immunofluorescent detection of two thymidine analogues (CldU and IdU) in primary tissue.
Immunofluorescent detection of two thymidine analogues (CldU and IdU) in primary tissue.
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DOI：
10.3791/2166
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发表时间：
2010-12-07
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影响因子：
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通讯作者：
Kushner, Jake A
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作者：
Tuttle, Alex H;Rankin, Matthew M;Kushner, Jake A
细胞分裂的精确测量是实验生物学中的一个基本挑战，当分析缓慢分裂的细胞时，这变得越来越复杂。已建立的检测细胞分裂的方法包括在细胞培养物中通过连续显微镜直接可视化、稀释活体染料如羧基荧光素二乙酸琥珀酰亚胺酯（CFSE）、免疫检测促有丝分裂抗原如ki 67或PCNA和胸苷类似物。胸苷类似物可通过多种方法检测，包括放射性检测氚化胸苷，免疫检测溴脱氧尿苷（BrdU）、氯脱氧尿苷（CldU）和碘脱氧尿苷（IdU），以及化学检测乙炔基脱氧尿苷（EdU）。我们已经推导出一种策略来检测不同的胸苷类似物（CldU和IdU）的成年小鼠的组织中的顺序掺入。我们的方法使研究人员能够准确地量化连续两轮的细胞分裂。通过优化免疫染色方案，我们的方法可以检测通过饮用水给药的非常低剂量的胸苷类似物，对小鼠长时间给药是安全的。因此，我们的技术可用于检测非常长寿的组织中的细胞更新。最佳免疫荧光染色结果可以在多种组织类型中实现，包括胰腺、皮肤、肠道、肝脏、肾上腺、睾丸、卵巢、甲状腺、淋巴结和脑。我们也应用这种技术来鉴定组织内的致癌转化。我们进一步应用这种技术来确定是否transit-amplifying细胞有助于组织的生长或更新。从这个意义上说，序贯给药的胸苷类似物代表了一种新的方法，用于研究参与组织稳态的细胞的起源和生存。
Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are analyzed. Established methods to detect cell division include direct visualization by continuous microscopy in cell culture, dilution of vital dyes such as carboxyfluorescein di-aetate succinimidyl ester (CFSE), immuno-detection of mitogenic antigens such as ki67 or PCNA, and thymidine analogues. Thymidine analogues can be detected by a variety of methods including radio-detection for tritiated thymidine, immuno-detection for bromo-deoxyuridine (BrdU), chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU), and chemical detection for ethinyl-deoxyuridine (EdU). We have derived a strategy to detect sequential incorporation of different thymidine analogues (CldU and IdU) into tissues of adult mice. Our method allows investigators to accurately quantify two successive rounds of cell division. By optimizing immunostaining protocols our approach can detect very low dose thymidine analogues administered via the drinking water, safe to administer to mice for prolonged periods of time. Consequently, our technique can be used to detect cell turnover in very long-lived tissues. Optimal immunofluoresent staining results can be achieved in multiple tissue types, including pancreas, skin, gut, liver, adrenal, testis, ovary, thyroid, lymph node, and brain. We have also applied this technique to identify oncogenic transformation within tissues. We have further applied this technique to determine if transit-amplifying cells contribute to growth or renewal of tissues. In this sense, sequential administration of thymidine analogues represents a novel approach for studying the origins and survival of cells involved in tissue homeostasis.