Gillitzer R, Berger R, Moll H
Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia.
J Histochem Cytochem. 1990 Mar;38(3):307-13. doi: 10.1177/38.3.1689333.
We have developed a reliable and sensitive immunohistochemical staining technique which allows the simultaneous demonstration of two different antigens expressed in or on the same cell (referred to as mixed labeling), together with the evaluation of the general histopathological appearance of the tissue. The staining procedure combines a three-step (streptavidin-biotin) immunogold-silver staining (IGSS) with a three-step immunoenzymatic labeling. For this purpose, we investigated the compatibility of IGSS with various substrates of peroxidase or alkaline phosphatase (AP). Highly reliable and discernible mixed labeling was achieved only after initial labeling with IGSS followed by AP labeling using the substrates naphthol AS-MX phosphate/Fast Blue or naphthol AS-BI phosphate/New Fuchsin, respectively. To ensure utmost specificity, we applied FITC-conjugated mouse monoclonal antibodies and rabbit anti-FITC immunoglobulins visualized by AP-labeled immunoglobulins and the respective substrate in a final step. This novel approach provides an excellent means for demonstration of immunocompetent cells and unequivocal determination of the percentage of specific cell subsets in infiltrated tissue. The advantages of this method, as compared with double immunofluorescence or double immunoenzymatic labeling, were investigated and are discussed.
我们开发了一种可靠且灵敏的免疫组织化学染色技术，该技术能够同时显示在同一细胞内或细胞表面表达的两种不同抗原（称为混合标记），同时还能评估组织的一般组织病理学外观。染色程序将三步（链霉亲和素-生物素）免疫金-银染色（IGSS）与三步免疫酶标记相结合。为此，我们研究了IGSS与各种过氧化物酶或碱性磷酸酶（AP）底物的兼容性。只有在用IGSS进行初始标记后，分别使用底物萘酚AS-MX磷酸酯/固蓝或萘酚AS-BI磷酸酯/新番红进行AP标记，才能实现高度可靠且可辨别的混合标记。为确保最大特异性，我们应用了异硫氰酸荧光素（FITC）偶联的小鼠单克隆抗体和兔抗FITC免疫球蛋白，在最后一步通过AP标记的免疫球蛋白和相应底物进行可视化。这种新方法为显示免疫活性细胞和明确确定浸润组织中特定细胞亚群的百分比提供了一种极好的手段。我们研究并讨论了该方法与双重免疫荧光或双重免疫酶标记相比的优势。
