Improvements in histological quality and signal retention following in situ hybridization in early chick embryos using plastic resin and recolorization.
Improvements in histological quality and signal retention following in situ hybridization in early chick embryos using plastic resin and recolorization.
复制标题
使用塑料树脂和重新着色对早期鸡胚进行原位杂交后，组织学质量和信号保留得到改善。
DOI：
10.1080/10520290500050957
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Schoenwolf,GC
中科院分区：
文献类型：
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作者：
Sawitzke,AL;Chapman,SC;Bleyl,SB;Schoenwolf,GC
我们描述了一种新的方法，可以在塑料嵌入胚胎的薄片中可靠地检测原位杂交信号。与石蜡、明胶、琼脂糖包埋切片或冷冻切片相比，塑料包埋的胚胎切片更薄，组织学质量更好；然而，由于信号的损失，塑料树脂传统上没有被用作原位杂交后的包埋介质。当用碱性磷酸酶和NBT/BCIP检测信号时，当样品暴露于有机化合物时，所得到的有色沉淀物会褪色。在塑料切片后，通过重复NBT/BCIP反应可使有色沉淀物重新沉积。这种再着色显示没有特异性损失，因为只有在抗地高辛/碱性磷酸酶偶联抗体与核糖探针结合的地方才检测到信号。强信号可以在不重新着色的情况下检测；然而，较弱的信号需要重新着色步骤。这种在处理和切片后重新沉积有色沉淀物的新方法可以准确确定基因表达的位置，并在高质量的早期鸡胚胎组织学切片中研究这种表达。
We describe a novel method that allows reliable detection of in situ hybridization signals in thin sections of plastic embedded embryos. Sections from plastic embedded embryos are thinner and have superior histological quality compared to paraffin, gelatin, agarose embedded sections or cryosections; however, plastic resin traditionally has not been used as an embedding medium following in situ hybridization because of loss of signal. When signal is detected with alkaline phosphatase and NBT/BCIP, the resulting colored precipitate is subject to fading when samples are exposed to organic compounds. The colored precipitate can be redeposited by repeating the NBT/BCIP reaction following plastic sectioning. This recolorization shows no loss of specificity, because signal is detected only where the anti-digoxigenin/alkaline phosphatase conjugated antibody is bound to the riboprobe. Strong signals can be detected without recolorization; however, weaker signals require the recolorization step. This novel method of re-depositing colored precipitate after processing and sectioning allows accurate determination of the location of gene expression and study of this expression in high quality histological sections of early chick embryos.