石蜡组织荧光原位杂交关键实验步骤探讨

目的：探讨石蜡组织荧光原位杂交（FISH）技术中关键实验步骤的最佳条件，以期提高石蜡切片FISH阳性细胞检出率和实验成功率。方法：在前期标本处理方面，进行双蒸水、0．5×SSC溶液、0．1％硫代亚硫酸钠溶液煮沸对比；在蛋白酶消化方面，设置胃蛋白酶和蛋白酶K两种消化方法，并在37℃条件下设置时间的梯度变化，比较其消化效果；对石蜡切片的变性设置温度梯度，比较杂交检出率；比较DAPI复染时不同浓度对单色、双色FISH结果的影响；应用抗淬灭剂后不同保存时间的对比。结果：采用0．5×SSC溶液煮沸15min，用200μg／mL蛋白酶K在37℃、6～10min条件下消化，可以取得较好的FISH效果；变性温度为81℃时检出率更高，DAPI复染浓度为1000ng／mL时针对单色FISH较合适，而浓度为500、150ng／mL时针对双色／多色FISH有较好的效果。结论：FISH条件经过对比得到优化，对石蜡组织FISH实验具有一定的指导意义。

Comparative Study on the Different Step of Fluorescence in Situ Hybridization in Paraffin Slides

Objective： To obtain the optimal methods of the fluorescence in situ hybridization（FISH）, for increas- ing success rate in FISH detection. Methods： There were three kinds of solution including water, 0.5×SSC buffer, 0.5% sodium thiosulfate solution, which were used to boil the slides, then comparing the experimenter effect with each other. The proteinase K and pepsin were adopted to digest the slides at 37℃, set up different time point to find the optimum results. About DNA denaturation of paraffin slides, we set different temperature to compared the detection rate. In addition, we also used different concentration of DAPI to staining the cell nucleus. To observe dyeing effect of nuclei, we used fluorescent quenches agent to confirm the preserved effect to clear how long the fluorescent is good. Results： The 0.5×SSC buffer for treating the slides was more effective solution. The proteinase K was an ideal digestive protein at 37℃, time 6-10 min, the digestion degreen was easy to control under this conditions. The denaturation temperatures as 81℃ would contribute to obtain a higher positive efficiency. After dyed with DAPI, the nucleus intact was in 1000 ng/mL showing clear blue. But for dual color and multiu-color FISH,the DAPI concentration should decrease. After application quenches agent, fluorescent signals saved good after 6 months, but capturing should perform in one mouths. Conclusion： The result of the optimized conditions of FISH is significantly better than that of common conditions, which is significant for improve the FISH results.