Owens Neil C, Hess F Martin, Badoer Emilio
School of Medical Sciences, RMIT University, Melbourne, Australia.
Methods Mol Biol. 2006;326:163-71. doi: 10.1385/1-59745-007-3:163.
A method is described for in situ hybridization of riboprobes to free-floating brain sections. Brain sections are hybridized and processed free-floating in buffer, i.e., without attachment to a support such as a slide. To withstand the extra wear compared with sections processed on-slide, the brain tissue must be well fixed (4% paraformaldehyde) and sections cut at thickness of typically 40 microm. Sections were exposed to a prehybridization treatment before a riboprobe is added to form the hybridization solution. Riboprobes were prepared from cDNA via an in vitro transcription reaction and are labeled with digoxigenin. The sections are subsequently processed to remove nonspecific binding and the digoxigenin label detected via an antibody conjugated to alkaline phosphatase. This method may be readily combined with neuronal tracing and is ideal for further processing by immunohistochemistry to detect specific proteins.
描述了一种将核糖核酸探针原位杂交到游离脑切片上的方法。脑切片在缓冲液中进行游离杂交和处理，即不附着在如载玻片等支持物上。为了承受与在载玻片上处理的切片相比额外的磨损，脑组织必须充分固定（4%多聚甲醛），切片厚度通常为40微米。在加入核糖核酸探针形成杂交溶液之前，切片要进行预杂交处理。核糖核酸探针通过体外转录反应从互补脱氧核糖核酸制备，并用地高辛标记。随后对切片进行处理以去除非特异性结合，并通过与碱性磷酸酶偶联的抗体检测地高辛标记。该方法可以很容易地与神经元追踪相结合，并且非常适合通过免疫组织化学进一步处理以检测特定蛋白质。
