Fixation/Permeabilization: New Alternative Procedure for Immunofluorescence and mRNA In Situ Hybridization of Vertebrate and Invertebrate Embryos
Fixation/Permeabilization: New Alternative Procedure for Immunofluorescence and mRNA In Situ Hybridization of Vertebrate and Invertebrate Embryos
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DOI：
10.1002/dvdy.23943
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发表时间：
2013-05-01
影响因子：
2.5
通讯作者：
Fuentes, Ricardo
中科院分区：
文献类型：
--
作者：
Fernandez, Juan;Fuentes, Ricardo
一个新的程序，描述了可视化的蛋白质和mRNA的表达在冷冻切片或整体安装水蛭，果蝇，斑马鱼和鸡胚胎的空间格局。我们的主要贡献是在使用一个非传统的固定/透化程序的基础上使用甲醛或多聚甲醛结合短C链羧酸。省略了去污剂、甲醇和蛋白酶。杂交程序是从常规使用的协议开发相同的胚胎修改。结果表明，细胞骨架和其他细胞质蛋白，以及不同的mRNA，在胚胎的预期区域清晰可见。我们的程序比目前使用的协议有几个优点：更简单，产生更好的一般保存细胞，产生可靠的结果，并可用于在不同发育阶段的不同类群的胚胎。据推测，短C链脂肪族羧酸调节醛固定剂对细胞蛋白质的交联作用。Developmental Dynamics 242：503-517，2013. (C)2013 Wiley Periodicals，Inc.
A new procedure is described to visualize the spatial pattern of expression of proteins and mRNAs in cryosections or whole-mounted leech, Drosophila, zebrafish, and chick embryos. Our principal contribution is in the use of a nonconventional fixation/permeabilization procedure based on the use of formaldehyde or paraformaldehyde combined with a short C-chain carboxylic acid. Detergents, methanol, and proteinases were omitted. Hybridization procedures were modified from those of routinely used protocols developed for the same embryos. Results showed that cytoskeletal and other cytoplasmic proteins, as well as different mRNAs, were clearly visualized in the expected regions of the embryos. Our procedure has several advantages over currently used protocols: is simpler, produces better general preservation of cells, yields reliable results, and can be used for embryos of different taxa at different developmental stages. It is hypothesized that short C-chain aliphatic carboxylic acids modulate the cross-linking effect of aldehyde fixatives on cell proteins. Developmental Dynamics 242:503-517, 2013. (C) 2013 Wiley Periodicals, Inc.