Improved preservation of X-gal reaction product for electron microscopy using hydroxypropyl methacrylate
Improved preservation of X-gal reaction product for electron microscopy using hydroxypropyl methacrylate
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DOI：
10.1016/j.neulet.2004.10.023
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发表时间：
2005-02-01
影响因子：
2.5
通讯作者：
Ono, K
中科院分区：
文献类型：
--
作者：
Masahira, N;Ding, L;Ono, K
在谱系追踪分析中，β -半乳糖苷酶（P-gal）基因是一种常用的报告基因，因为它在5-溴-4-氯-3-吲哚- β - d -半乳糖苷（X-gal）的组织化学检测中相对稳定且高度敏感。明确确定标记细胞的类型和特征需要透射电子显微镜（TEM）检查其形态。X-gal染色是由P-gal和X-gal反应形成的沉淀，在光镜下通常被认为是浅蓝色或绿色的反应产物。然而，TEM制备的标准方案削弱了用环氧丙烷取代乙醇的反应强度或导致X-gal反应产物的损失。为了解决这个问题，我们证明甲基丙烯酸羟丙酯可以很好地保存X-gal反应产物。本文提出的方案似乎对所有类型的x -gal染色组织的TEM谱系测定是有用的。2004爱思唯尔爱尔兰有限公司版权所有。
In lineage tracing analysis, the beta-galactosidase (P-gal) gene is a commonly used as a reporter gene because it is relatively stable and highly sensitive in histochemical detection using 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal). Clear determination of the types and characteristics of labeled cells requires transmission electron microscopic (TEM) examination of their morphology. X-gal staining, which involves the precipitate formed by the reaction between P-gal and X-gal, is usually recognized as a light blue or green reaction product on light microscopic (LM) examination. However, the standard protocol for TEM preparation weakens the intensity of or results in the loss of X-gal reaction product at the step of substitution of ethanol with Epon using propylene oxide. To solve this problem, we show that hydroxypropyl methacrylate achieves good preservation of X-gal reaction products. The protocol presented here appears to be useful for lineage determination by TEM of all types of X-gal-stained tissues. (C) 2004 Elsevier Ireland Ltd. All rights reserved.