Speel E J, Saremaslani P, Roth J, Hopman A H, Komminoth P
Department of Pathology, University of Zürich, Switzerland.
Histochem Cell Biol. 1998 Dec;110(6):571-7. doi: 10.1007/s004180050319.
We report an optimized in situ hybridization (ISH) protocol with a rapid signal amplification procedure based on catalyzed reporter deposition (CARD) to increase the sensitivity of non-isotopic mRNA ISH on formaldehyde-fixed and paraffin-embedded tissue. The CARD method is based on the deposition of haptenized tyramide molecules in the vicinity of hybridized probes catalyzed by horseradish peroxidase. Commercially available and newly synthesized haptenized tyramides, including digoxigenin-, biotin-, di- and trinitrophenyl- as well as fluorescein-tyramide, were compared. The haptenized tyramides were visualized using peroxidase conjugated anti-hapten antibodies followed by the diaminobenzidine reaction. As a test system, we applied digoxigenin-labeled oligonucleotides to detect insulin and vasoactive intestinal polypeptide mRNA in pancreatic endocrine tumors and liver metastases. Our results indicate that specificity, sensitivity, and applicability of oligonucleotide mRNA ISH can be significantly improved by using chemically digoxigenin-labeled oligonucleotide probes and signal amplification by CARD. Furthermore, all tested tyramides provided approximately equal amplification efficiency. In conclusion, CARD signal amplification should further promote mRNA ISH studies on paraffin-embedded tissues and allow for multiple-target nucleic acid detection in situ.
我们报告了一种优化的原位杂交（ISH）方案，该方案采用基于催化报告分子沉积（CARD）的快速信号放大程序，以提高在甲醛固定石蜡包埋组织上进行的非同位素mRNA原位杂交的灵敏度。CARD方法基于辣根过氧化物酶催化在杂交探针附近沉积半抗原化的酪胺分子。我们比较了市售的和新合成的半抗原化酪胺，包括地高辛、生物素、二硝基苯基和三硝基苯基以及荧光素酪胺。使用与过氧化物酶偶联的抗半抗原抗体，随后进行二氨基联苯胺反应来观察半抗原化酪胺。作为测试系统，我们应用地高辛标记的寡核苷酸来检测胰腺内分泌肿瘤和肝转移灶中的胰岛素和血管活性肠多肽mRNA。我们的结果表明，通过使用化学地高辛标记的寡核苷酸探针和CARD信号放大，可以显著提高寡核苷酸mRNA原位杂交的特异性、灵敏度和适用性。此外，所有测试的酪胺提供了大致相同的放大效率。总之，CARD信号放大应进一步推动石蜡包埋组织的mRNA原位杂交研究，并允许原位进行多靶点核酸检测。
