E8.5-E12.5大鼠胚胎整体原位杂交技术

Whole mount in situ hybridization of E8.5 to E12.5 rat embryos

目的:通过优化实验条件,建立大鼠胚胎整体原位杂交方法。方法:E8.5-E10.5大鼠胚胎4%多聚甲醛固定6 h,不经蛋白酶K消化;E11.5-E12.5胚胎4%多聚甲醛固定过夜,蛋白酶K系列稀释实验确定消化浓度和时间。E8.5-E12.5胚胎预杂交2 h,地高辛标记的甘油二酯激酶ζ(DGKζ)寡核苷酸探针杂交24 h,山羊血清封闭2.5～3 h,地高辛抗体4℃过夜,冷TBST和NTMT漂洗1～2 h,NBT/BCIP避光显色。应用免疫组织化学方法观察DGKζ在E12.5大鼠胚胎组织的表达。结果:DGKζ在不同胚龄大鼠胚胎均有清晰的杂交信号,主要集中在神经系统;E12.5大鼠胚胎脑泡、背根神经节有较强表达,与免疫组化结果一致。结论:本研究建立了简便、可行、重复性高的E8.5-E12.5大鼠胚胎整体原位杂交方法。

Objective： To establish a method for whole mount in situ hybridization of rat embryos by optimizing experimental conditions. Methods： E8.5-El0.5 rat embryos were fixed in 4% paraformaldehyde for 6 hours, and had no proteinase K digestion. E11.5-E12.5 rat embryos were fixed in 4% paraformaldehyde overnight. The concentration and time of proteinase K digestion were analyzed by serial dilution. ES. 5-E12.5 embryos were pre-hybridized for 2 hours and hybridized with digoxigenin-labeled diaeylglycerol kinase ζ （DGKζ）oligonucleotide probe for 24 hours, blocked by goat serum for 2.5-3 hours, and incubated anti-digoxigenin antibody overnight at 4℃. Then the embryos were rinsed in cool TBST and NTMT for 1-2 hours and developed with NBT/BCIP in dark. The expression of DGKζ protein in E12.5 embryos was observed by using immunohistoehemistry. Results： DGKζ positive signals mainly located in nervous system of rat embryos at different ages and were viewed obviously. DGKζ positive signals were observed strongly in cephalic vesicles, dorsal root ganglions of E12.5 rat embryos, which was consistent with the immunohistochemical results. Conclusion： The present study has established a simple, reliable and reproducible method for whole mount in situ hybridization of E8.5 to E12.5 rat embryos.