应用直接酪胺信号放大方法显示Y染色体荧光原位杂交信号

Detection of the Signal from the Mouse Y Chromosome in Situ Hybridization with Direct Tyramine Signal Amplification

目的用直接酪胺信号放大(tyramine signal amplification,TSA)方法显示Y染色体荧光原位杂交信号,以提高检测脑组织切片Y染色体荧光原位杂交信号的灵敏度。方法以小鼠Y染色体重复序列pY353/B cDNA为模板标记RNA探针,以雌性小鼠为对照,用原位杂交方法杂交Y染色体特异性基因区域,分别用直接TSA方法和"三步抗体法"显示荧光原位杂交信号。通过计数Y染色体阳性细胞核占所有细胞核的百分率来计算TSA法和"三步抗体法"检测脑组织切片Y染色体荧光原位杂交信号的灵敏度,比较分析两种方法灵敏度的差异。结果"三步抗体法"在脑切片检测Y染色体阳性信号的灵敏度和特异度分别为85.67%和100%;直接TSA法在脑切片检测Y染色体阳性信号的灵敏度和特异度分别为92.82%和100%。结论TSA方法检测脑组织切片Y染色体荧光原位杂交信号的灵敏度比"三步抗体"法有显著的提高,可以用于脑内追踪和分析骨髓干细胞的迁移规律。

Objective To enhance the sensitivity of detecting the signal from mouse Y chromosome fluorescence in situ hybridization of brain slices direct tvramine signal amplification method was applied. Methods The RNA probe was labeled with pY353/ B, a repeat sequence of mouse Y chromosome. Compared with the female mice, the brain tissue sections of the male mice were in situ hybridized with the RNA probe, its fluorescence signal was detected with direct tyramine signal amplification method and ＂three antibodies method＂ respectively. The sensitivity of the detection Y chromosome positive cells was calculated as the percentage of the Y chromosome positive cell nuclei to that of all nuclei. The difference of sensitivity between the direct tyramine signal amplification and ＂three antibodies method＂ was compared and analyzed with statistic methods. Results The sensitivity and specificity of ＂three antibodies method＂ for detection of Y chromosome signal in brain tissue is 85.67% and 100% , respectively; the sensitivity and specificity of direct tyramine signal amplification method for detection of Y chromosome signal in brain tissue is 92.82% and 100% , respectively. Conclusion Compared with the ＂three antibodies method＂, the sensitivity for detection of the signal of Y chromosome fluorescence in situ hybridization in brain tissue sections of mice was enhanced significantly by the direct tyramine signal amplification method. This technique can be used in the tracing and analysis of the migration of bone marrow stem cells in the brain.