(二)基本操作方法(Fischer D et al, 1992) 1.组织处理 (1)固定:固定剂采用4%多聚甲醛(PFA,用PBS配制)加0.5%戊二醛(GA),保存在4℃。作者采取浸入法,即取大鼠肝脏放入冷固定剂中(4%PFA,不含GA),切成1mm3左右的小方块。修整好的组织块移入另一玻皿中,内盛有4%PFA和0.5%GA,固定2h。 (2)PBS(4℃)漂洗3~5min。 (3)脱水 时间乙醇浓度温度 2×15min 30% 4℃ 30min 30% 4℃ 30min 50% -20℃ 2×30min 70%,90%,96%,100% -20℃ 注意勿使酒精挥发致使组织干燥。 (4)浸透和包埋(Carlemalarm et al, 1989):由于K4M包埋剂有挥发性,因此,此步操作者必须带手套,在通风柜中进行,注意K4M包埋剂不要靠近O2,一切在低温下进行,最好设有恒低温的冷柜。 ①Lowcryl K4M配方 1)交联剂A 2g 2)单体B 13g 3)引发剂 0.075g 混合1),2)后,再加“3)”轻搅匀,混匀后置于-20℃。 ②浸透(infiltration),在-20℃进行 时间液体 1h 100%乙醇:K4M(1:1,V/V) 2×1h K4M 过夜 K4M 2×3h K4M ③包埋: 先将胶囊冷却,滴入几滴K4M,然后每个胶囊内放入一个组织块,以K4M充满胶囊,在室温放置30min,使包埋剂中气泡逸出。然后置于0℃-40℃之间,利用紫外线灯360nm波长照射5天,温度必须保存恒定。然后移至室温使温度回升,胶囊变硬。 ④切片:由于胶囊是透明的,包埋在内的肝组织很容易识别。修块后进行切片,由于K4M是亲水性的,在切片过程中注意组织块表面一定不要让水浸湿,用200个网眼的镍网覆以For-mvar膜和碳膜捞取切片,空气干燥备用于杂交。 2.探针准备rRNA探针以Dig-UTP标记,注意探针不宜过长,否则影响对组织的穿透性。如过长,可事先用第二十章 介绍的探针水解法处理。 3.杂交本步骤均在湿盒内进行,将杂交液滴滴于蜡膜上,将镍网载有切片面覆于液滴上,在65℃杂交至少3h。杂交液含:5×SSC,0.1mg/ml tRNA,Dig-UTP反意探针10ng/μl(et 1×SSC配,含150mmol/L NaCl, 15mmol/L醋酸钠)。 4.杂交后漂洗 在室温用2×SSC漂洗3×5min,然后用PBST(PBS,0.1%Tween)漂洗2×10min。 5.显示 ①以PBST加BT封闭(BT配方:PBST,1%BSA)孵育15min。 ②应用抗地高辛抗体结合直径1mm的金粒,以PBST加BG稀释为1:30,室温孵育1h。 ③以PBST漂洗3×5min。 ④以带笔尖嘴软塑料壶冲洗6×15min。 ⑤以银增强法,在暗室中孵育于显影液:促进液(Enhancer)1:1,4~20min。 ⑥重复④。 ⑦以2%醋酸铀(4min),枸缘酸铅1min染色,漂洗,空气干燥。 ⑧电镜观察。 四、结语 原位分子杂交技术在电镜水平的应用,或简称为电镜原位分子杂交技术是基因表达在超微结构定位的一项极有前景的新兴技术。但要使之完善,还需要做许多工作。必须说明的是电镜原位杂交技术和光镜原位杂交技术一样必须设置对照实验组,对显示的结果的解释应持审慎的态度。一般应在重复多次实验的基础上才能得出对本实验的结论,不能只凭一次实验或一张电镜照片就勿忙结论。因原位杂交技术是高度敏感、高度特异性技术,影响因素很多。电镜原位杂交技术的影响和干扰因素更多。相信在不久的将来,随着电镜原位杂交技术的广泛应用,科技工作者将从实践中对本技术的实验流程不断完善并有更多的新的电镜原位杂交技术方法涌现。 参考文献 1.Bhatt B and MeGee JOD. Chromsomal assignment of gene. In:In situ hybridization, Principles and practice (Eds:Polak JM and McGee JOD)OxFord Science Publication, 1991:149~164 2.Hopman AHN et al.Interphase cytogenetics of solid tumors. In:In situ hybridisation, principles and practice (Eds:Polak JM and McGee JOD)OxFord Science Pulication, 1991:165~186 3.West JD.Sexing the human conceptus by in situ hybridization. In:In situ hybridisation, application to developmental biology and medicine (Eds:Harris N and Wilkinson DG).Combridge University Press, 1990:205~240 4.Koch J,et al. Oligonucleotide –priming methods for the chromosome –specific labelling of alpha satellite DNA in situ.Chromosome, 1989, 98:259~265 5.Evani Viegas –Pequignot et al.Mapping of single – copy DNA sequences on human chromsomes by in situ hybridisation with biotinylated probes:Enhancement of detection sensitivity by intensifield - fluorescence digital –image mieroscopy. Proc Natl Acad Sci USA. 1989,86:582~586 6.Mclaren A. Prenatal diagnosis before implantation:opportunities and problems. Prenatal Diagnosis, 1985;5:85~90 7.Julen JC, et al. Rapid prenatal diagnosis of Down’s syndrome with in situ hybridisation of fluorescent DNA probe. Lancet , Ⅲ:863~864 8.Millar DS, ET AL. Normal blood cell values in the early and –trimester fetus:Prenatal Diagnosis, 1985, 5:367~373 9.Buggers JD, et al. The culture of mouse embryos in vitro. In :Methods in mammalian Embryology, (Eds:Daniel JC Jr)San Francisco:Free man , 1971:86~116 10.Hutchison N et al. In situ hybridisation at the electron microscope level:Hybrid detection by autoradiography and colloidal gold, The J Cell Biology, 1982, 95:609~618 11. Binder M, et al. In situ hybridisation of the eletron microscope level:Localization of transcripts on ultrathin sections of Lowicryl K4M-embedded tissue using biotinylated probes and Protein A-Gold Complexes. J. Cell Biology, 1986, 102:1646~1653 12.Roth JM, et al. Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue. j Histochem. Cytochem, 1981,29:663~667 13.Lawrence LB and Singer RH.Quantitative analysis of in situ hybridisation methods for the detection of actin gene expression ,Nucleic Acids Res, 1985, 13:1777~1799 14.Weketer H deF,et al. Use of a biotinylated probe and in situ hybridisation for light and electron microscopic localization of P0 mRNAin myelin –forming Schwann cells, Histochemistry, 1987, 86:441~444 15.Carlsmalarm E and Villger W. Low temperature embedding. In:Tehcniques in immunocytochemistry (Eds Bullock GR and Petruoz P),Acad Press, 1989,4:29~44 16.焦仁杰,等.应用电镜原位分子杂交技术对腺病毒DNA在宿主细胞内进行定位.电子显微学报,1992,2:81~84 (蔡文琴) (责任编辑:泉水) |