van Vlierberghe Ronald L P, Sandel Maro H, Prins Frans A, van Iersel Liselot B J, van de Velde Cornelis J H, Tollenaar Rob A E M, Kuppen Peter J K
Department of Surgery, Leiden University Medical Centre, Leiden, the Netherlands.
Microsc Res Tech. 2005 May;67(1):15-21. doi: 10.1002/jemt.20181.
Immune-cell infiltration is frequently seen within human solid tumors. A detailed phenotypic analysis of these cells may aid in the understanding of an antitumor immune response. Standard hematoxylin/eosin and conventional immunohistochemical stainings are helpful, but have major limitations in the number of markers that can be identified and localized per tissue section. Therefore, we developed a combined fluorescence and brightfield microscopic technique by using both immunofluorescence and immunogold-silver methods, thereby discriminating three different leukocyte markers plus one tumor marker simultaneously in a single section. This enabled us to study both phenotype and location of infiltrating immune cells in colorectal tumors. We used a two-step staining in which primary and secondary antibodies were selected for minimal cross-reactivity. Furthermore, the secondary fluorescent antibody conjugates were selected for minimal spectral overlap. For computer-assisted analysis the brightfield microscopy image was combined with the fluorescence microscopy images. This combination of techniques provides a powerful tool for detailed multiparameter microscopic analysis of formalin-fixed, paraffin-embedded tissue sections in general and for tumor-immune cell infiltration in particular.
免疫细胞浸润在人类实体瘤中很常见。对这些细胞进行详细的表型分析可能有助于理解抗肿瘤免疫反应。标准苏木精/伊红染色和传统免疫组织化学染色很有用，但在每个组织切片中可识别和定位的标志物数量方面存在重大局限性。因此，我们开发了一种结合荧光和明场显微镜技术，同时使用免疫荧光和免疫金银法，从而在单个切片中同时区分三种不同的白细胞标志物和一种肿瘤标志物。这使我们能够研究结直肠癌中浸润免疫细胞的表型和位置。我们采用两步染色法，选择交叉反应最小的一抗和二抗。此外，选择光谱重叠最小的二抗荧光抗体偶联物。为了进行计算机辅助分析，将明场显微镜图像与荧光显微镜图像相结合。这种技术组合为一般的福尔马林固定、石蜡包埋组织切片，特别是肿瘤免疫细胞浸润的详细多参数显微镜分析提供了一个强大的工具。
