小鼠胚胎整体原位杂交实用技术的建立  

ESTABLISHMENT OF WHOLE-MOUNT IN SITU HYBRIDIZATION TO MOUSE EMBRYOS

作　　者：吴燕[1] 马子敏[1] 刘淑红[1] 范文红[1] 范明[1] 

机构地区：[1]军事医学科学院基础医学研究所脑保护与可塑性研究实验室,北京100850

出　　处：《解剖学报》2004年第4期434-436,共3页Acta Anatomica Sinica

基　　金：国家 8 63重大专项基金资助项目 ( 2 0 0 2BA711A0 1 0 3 )

摘　　要：目的　优化小鼠胚胎整体原位杂交及数据分析方法。　方法　小鼠胚胎用 4 %多聚甲醛 4℃固定过夜 ,10mg L蛋白酶K(PK)室温消化 30min ,预杂交 2h后 ,加入探针 6 3℃反应 16～ 18h ,用盐溶液高温洗涤多次 ,RNA酶 37℃消化 1h ,封闭 4h后加入地高辛抗体 4℃过夜。抗体反应后反复洗涤并在水平摇床上振摇 ,NBT和BCIP显色系统避光显色。提取图像灰度值 ,扣除背景、对照等假阳性信号。　结果　KIAA170 8探针在脑、脊髓等 8个区域有杂交信号 ,对图像数据分析结果表明 ,只有 1个区域的信号在 95 %可信限范围内 ,为阳性信号。　结论　通过控制蛋白酶K消化时间、探针和地高辛抗体浓度 ,以及洗涤、显色过程 ,得到了低本底、着色清晰的杂交结果 ,结合图像的数据分析步骤 ,消除假阳性结果。建立了一套实用的小鼠胚胎整体原位杂交方法。Objective To optimize the method of whole mount in situ hybridization of mouse embryos. Methods Mouse embryos were fixed in 4% paraformaldehyde in DEPC-PBS overnight at 4℃. After treated with 10*!mg/L proteinase K for 30 min,the embryos were pre-hybridized for 2-hours and hybridized with probes overnight at 63℃. The embryos were then digested with 20*!mg/L RNase A for 30 min and blocked for 4 hrs at room temperature.The embryos were incubated with anti-digoxigenin antibody overnight at 4℃ and developed with 4.5*!μl NBT and 3.5*!μl BCIP per ml of alkaline phosphatase buffer in dark for 2-20 hours.Results KIAA1708 positive signals were viewed in brain and spinal cord and other 6 regions. Only 1 region was KIAA1708 positive statistically. Conclusion Through optimizing several conditions which include concentration of probe and antibody,time of proteinase K digestion,the washing procedures,data assaying,we have established a practical method of mouse embryo whole mount in situ hybridization successfully.

关 键 词：方法改进 整体原位杂交 小鼠胚胎 

分 类 号：R329-34[医药卫生—人体解剖和组织胚胎学]