Todorović-Raković Nataša
Department of Experimental Oncology, Institute for Oncology and Radiology of Serbia, Belgrade, Serbia.
Methods Mol Biol. 2013;1012:249-54. doi: 10.1007/978-1-62703-429-6_17.
In situ hybridization (ISH) allows evaluation of genetic abnormalities, such as changes in chromosome number, chromosome translocations or gene amplifications, by hybridization of tagged DNA (or RNA) probes with complementary DNA (or RNA) sequences in interphase nuclei of target tissue. However, chromogenic in situ hybridization (CISH) is also applicable to formalin-fixed, paraffin-embedded (FFPE) tissues, besides metaphase chromosome spreads. CISH is similar to fluorescent in situ hybridization (FISH) regarding pretreatments and hybridization protocols but differs in the way of visualization. Indeed, CISH signal detection is similar to that used in immunohistochemistry, making use of a peroxidase-based chromogenic reaction instead of fluorescent dyes. In particular, tagged DNA probes are indirectly detected using an enzyme-conjugated antibody targeting the tags. The enzymatic reaction of the chromogenic substrate leads to the formation of strong permanent brown signals that can be visualized by bright-field microscopy at 40 × magnification. The advantage of CISH is that it allows the simultaneous observation of gene amplification and tissue morphology and the slides can be stored for a long time.
原位杂交(ISH)通过将标记的DNA(或RNA)探针与靶组织间期核中的互补DNA(或RNA)序列杂交,能够评估遗传异常情况,如染色体数目变化、染色体易位或基因扩增。然而,除了中期染色体铺展外,显色原位杂交(CISH)也适用于福尔马林固定、石蜡包埋(FFPE)组织。CISH在预处理和杂交方案方面与荧光原位杂交(FISH)相似,但在可视化方式上有所不同。实际上,CISH信号检测类似于免疫组织化学中使用的方法,利用基于过氧化物酶的显色反应而非荧光染料。具体而言,标记的DNA探针通过靶向标记的酶联抗体进行间接检测。显色底物的酶促反应会产生强烈的永久性棕色信号,可在40倍放大倍数下通过明场显微镜观察到。CISH的优点在于它能够同时观察基因扩增和组织形态,并且玻片可以长期保存。
J Cancer Res Clin Oncol. 2014-12