In situ hybridization with polymerase chain reaction-derived single-stranded DNA probe and S1 nuclease
In situ hybridization with polymerase chain reaction-derived single-stranded DNA probe and S1 nuclease
复制标题
DOI：
10.1007/s004180050327
复制
发表时间：
1999-01-01
影响因子：
2.3
通讯作者：
Maeda, S
中科院分区：
文献类型：
--
作者：
Kitazawa, S;Kitazawa, R;Maeda, S
一个快速和简化的协议，原位杂交（ISH）与聚合酶链反应（PCR）衍生的单链DNA探针和S1核酸酶显示的成绩单脱钙骨标本的骨基质蛋白。小鼠骨组织用4%多聚甲醛固定，用20%EDTA脱钙，并包埋在石蜡中。每对引物分别从小鼠骨连接素（ON）成熟蛋白编码区和小鼠骨桥蛋白（OP）成熟蛋白编码区扩增出280 bp和320 bp的DNA片段。洗脱、纯化初始PCR产物，并在地高辛（DIG）标记的dUTP存在下通过单向PCR再扩增。ISH通过蛋白酶K处理、杂交和洗涤进行。通过S1核酸酶处理选择性地去除未杂交的单链DNA探针。用碱性磷酸酶偶联的抗DIG抗体观察杂交的探针。在脱钙骨的薄切片上可清楚地检测到ON和OP的转录本。由于该方案不需要克隆或体外转录，因此可以在配备热循环仪的普通实验室中进行可靠和稳定的ISH。
A rapid and simplified protocol for in situ hybridization (ISH) with polymerase chain reaction (PCR)derived single-stranded DNA probes and S1 nuclease revealed transcripts of bone matrix proteins on decalcified skeletal bone specimens. Mouse bone tissue was fixed with 4% paraformaldehyde, decalcified with 20% EDTA, and embedded in paraffin. Each pair of primers for reverse transcriptase -PCR was designed to amplify a 280-bp DNA fragment from the coding region of the mature protein of mouse osteonectin (ON) and a 320-bp fragment from the coding region of mouse osteopontin (OP). Initial PCR products were eluted, purified, and reamplified by unidirectional PCR in the presence of the digoxigenin (DIG)-labeled dUTP. ISH was carried out by proteinase K treatment, hybridization, and washing. The unhybridized single-stranded DNA probe was selectively removed by S1 nuclease treatment. Hybridized probes were visualized with the alkaline phosphatase-conjugated anti-DIG antibody. The transcripts of ON and OP were clearly detected on the thin sections of the decalcified bone. Because this protocol does not require cloning or in vitro transcription, reliable and stable ISH can be done in an ordinary laboratory equipped with a thermal cycler.