Mullink H, Vos W, Jiwa M, Horstman A, van der Valk P, Walboomers J M, Meijer C J
Institute of Pathology, Free University Hospital, Amsterdam, The Netherlands.
J Histochem Cytochem. 1992 Apr;40(4):495-504. doi: 10.1177/40.4.1532404.
Silver-intensification methods described in the literature for the diaminobenzidine (DAB) and diaminobenzidine-nickel (DAB/Ni) endproduct of the peroxidase reaction were compared in model systems after immunoperoxidase and in situ hybridization. First, these methods were compared in immunohistochemical model systems, using the demonstration of glial fibrillar acidic protein (GFAP) and prostate-specific antigen (PSA) in paraffin sections of human brain and prostate tissue, respectively. When DAB without Ni was used as substrate, tissue argyrophilia caused considerable background staining. Only when this tissue reactivity was quenched with, e.g., CuSO4 with H2O2 or thioglycolic acid, were the results acceptable. A considerable improvement in the signal-to-noise ratio could be obtained when nickel was included in the substrate mixture. The methods that proved to be best for demonstration of GFAP and PSA made use of acid developer solutions. Subsequently, these methods were compared with other sensitive immunostaining methods for demonstration of the gamma-delta T-cell receptor in frozen lymphoid tissue. In this model a considerable increase in the number of positive cells could be obtained using silver intensification. The different methods using DAB/Ni were also compared for use in DNA in situ hybridization (DISH). In this case two model systems were used: human papilloma virus type 11 (HPV-11) DNA in condyloma tissue (abundant target model) and Epstein-Barr virus (EBV) DNA in a mononucleosis lymph node (low target model). For demonstration of HPV-11, all methods gave more or less satisfactory results, which were best with the acid developer solutions. Moreover, for demonstration of EBV DNA, a signal could be obtained only with these developer solutions. Such a method also proved suitable in double immuno-hybrido stainings for the demonstration of EBV DNA in specific antigen-positive Reed-Sternberg cells in paraffin sections of Hodgkin lymph nodes.
在免疫过氧化物酶和原位杂交后的模型系统中，对文献中描述的用于过氧化物酶反应的二氨基联苯胺（DAB）和二氨基联苯胺-镍（DAB/Ni）终产物的银强化方法进行了比较。首先，在免疫组织化学模型系统中对这些方法进行了比较，分别在人脑和前列腺组织的石蜡切片中使用胶质纤维酸性蛋白（GFAP）和前列腺特异性抗原（PSA）的显色。当不添加镍的DAB用作底物时，组织嗜银性会导致相当多的背景染色。只有当用例如硫酸铜与过氧化氢或巯基乙酸淬灭这种组织反应性时，结果才可以接受。当底物混合物中加入镍时，可以显著提高信噪比。被证明对GFAP和PSA显色效果最佳的方法使用了酸性显影剂溶液。随后，将这些方法与用于在冷冻淋巴组织中显示γ-δ T细胞受体的其他敏感免疫染色方法进行了比较。在这个模型中，使用银强化可以使阳性细胞数量显著增加。还比较了使用DAB/Ni的不同方法在DNA原位杂交（DISH）中的应用。在这种情况下，使用了两个模型系统：尖锐湿疣组织中的人乳头瘤病毒11型（HPV-11）DNA（丰富靶标模型）和单核细胞增多症淋巴结中的爱泼斯坦-巴尔病毒（EBV）DNA（低靶标模型）。对于HPV-11的显色，所有方法都或多或少给出了令人满意的结果，酸性显影剂溶液的效果最佳。此外，对于EBV DNA的显色，只有使用这些显影剂溶液才能获得信号。这样的方法也被证明适用于双重免疫杂交染色，用于在霍奇金淋巴瘤石蜡切片中特定抗原阳性的里德-斯腾伯格细胞中显示EBV DNA。
