Multiple immunoenzyme staining: Methods and visualizations for the observation with spectral imaging
Multiple immunoenzyme staining: Methods and visualizations for the observation with spectral imaging
复制标题
DOI：
10.1369/jhc.2007.950170
复制
发表时间：
2008-04-01
影响因子：
3.2
通讯作者：
van der Loos, Chris M.
中科院分区：
文献类型：
--
作者：
van der Loos, Chris M.
存在几种染色概念和颜色组合，以在人体组织标本上成功进行双重免疫酶染色。这些概念大多基于两种一抗之间的差异：动物种属、小鼠IG同种型或IgG亚类、缀合物或浓度。传统上，双重免疫酶染色使用选择的色原，以在用肉眼观察时提供最大的颜色对比度。不幸的是，视觉上良好的颜色组合总是包括至少一个漫射色原，因为缺乏适当的色原颜色。这种情况是彻底改变了光谱成像的使用，其中显微镜可以在单独的图像中基于它们的光谱特性进行分解。光谱分解甚至可以进行四重免疫酶染色。这项工作包含了一些常见的一抗组合的免疫酶双染色程序的实用建议：兔-小鼠，山羊-小鼠，小鼠-小鼠，兔-兔。建议的方案都适用于经典的红棕色组合加蓝色核复染，其分别由过氧化物酶活性（二氨基联苯胺四盐酸盐）、碱性磷酸酶活性（液体永固红）和苏木精组成。虽然红色和棕色的色原在视觉上没有很好的对比，但它们都显示出清晰的定位，并且可以通过光谱成像完全分离。
Several staining concepts and color combinations exist to perform successful double immunoenzyme staining on human tissue specimens. Most of these concepts are based on differences between both primary antibodies: animal species, mouse Ig isotype or IgG subclasses, conjugates, or concentrations. Traditionally, double immunoenzyme staining has used chromogens selected to provide maximum color contrast when observed with the unaided eye. Unfortunately, visually good color combinations always include at least one diffuse chromogen, because of the paucity of appropriate chromogen colors. This situation is drastically changed with the use of spectral imaging, where multicolor microscopy can be unmixed in individual images based on their spectral characteristics. Spectral unmixing can be performed even up to quadruple immunoenzyme staining. This work contains practical suggestions for immunoenzyme double staining procedures for some frequently encountered primary antibody combinations: rabbit-mouse, goat-mouse, mouse-mouse, and rabbit-rabbit. The suggested protocols are all suitable for a classical red-brown color combination plus blue nuclear counterstain that is composed of peroxidase activity (diaminobenzidine tetra hydrochloride), alkaline phosphatase activity (Liquid Permanent Red), and hematoxylin, respectively. Although the red and brown chromogens do not contrast very well visually, they both show a crisp localization and can be perfectly unmixed by spectral imaging.