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由于同位素基因探针原位杂交组化实验周期长,而且对人体有害,因此人们一直希望获得一种非周位素基因探针,而其敏感性与同位素基因探针相似。本文介绍一种光敏生物素标记的反意SScRNA探针显示组织切片SSmRNA的方法,其敏感性与我们以前介绍的~3H-cRNA探针相似。一、材料与方法:含前生长抑素原基因的质粒来源、性质及质粒的线性化请参考我们以前的报导。线性化后,以线性质粒DNA为模板合成未加任何标记物的反意SScRNA。纯化,乙醇沉淀回收此cRNA后,将其溶于灭菌双蒸水中,使其终浓度为0.5-1.0μg/μ1,再与等体积的光敏生物素(1μg/μl)混合,在150瓦卤素灯下,距光源20cm处照射30分钟。用仲丁醇抽提游离生物素,最后丁醇沉淀回收光敏生物素反意SSc-RNA探针,并将其溶于适量的灭菌双蒸水,用紫外分光光度计测探针的OD_260,计算探针浓度。切片的制作,预处理、预杂交、杂交、杂交后冲洗详见我们以前介绍的方法,但探针浓度不同,其最佳工作浓度为2μg/ml,每一切片覆盖20μ1。切片彻底冲洗后,3%牛血清蛋白,37℃孵育1小时,亲和素碱性磷酸酶(1∶500)25℃孵育2小时,然后分别用TSM_1(0.1MTris-Hcl PH7.5、1M Nacl,2mMMgcl_2)冲洗20分钟
Because isotope gene probes hybridize in situ for long periods of histochemical staining and are harmful to humans, one has always wanted to obtain a non-isotope gene probe that is similar in sensitivity to isotopic gene probes. This article presents a photo biotin-labeled anti-SScRNA probe showing tissue-sectioned SSmRNA with similar sensitivity to the ~ 3H-cRNA probe we previously described. I. MATERIALS AND METHODS: The source, nature and plasmid linearization of the pre-somatostatin-containing gene is described in our previous report. After linearization, linearized plasmid DNA was used as a template to synthesize anti-SScRNA without any marker. Purification, Ethanol precipitation After recovering this cRNA, it was dissolved in sterilized double-distilled water at a final concentration of 0.5-1.0 μg / μl and mixed with an equal volume of biotin (1 μg / μl) Under the lamp, 20cm away from the light source for 30 minutes. The free biotin was extracted with sec-butanol, and finally the butanol was precipitated to recover the photosensitive biotin anti-sense SSc-RNA probe, which was dissolved in an appropriate amount of sterilized double-distilled water. The probe OD260 was measured with a UV spectrophotometer, Calculate the probe concentration. The preparation, pre-treatment, pre-hybridization, hybridization and post-hybridization rinses are described in detail in our previous section. However, the best working concentrations for the probes are 2 μg / ml and 20 μl for each section. After thorough washing, 3% bovine serum albumin was incubated for 1 hour at 37 ° C and for 2 hours at 25 ° C with avidin alkaline phosphatase (1: 500) and then incubated with TSM 1 (0.1M Tris-HCl pH 7.5, 1 M Nacl , 2 mM MgCl 2) for 20 minutes