Hsu S M, Soban E
J Histochem Cytochem. 1982 Oct;30(10):1079-82. doi: 10.1177/30.10.6182185.
Three metallic ions, NiCl2, CoCl2, and CuSO4, were found to modify the color of the normally brown diaminobenzidine (DAB) reaction. The colors ranged from purplish blue (NiCl2), dark blue/bluish black (CoCl2), to greyish blue (CuSO4). We have found that the CoCl2 + DAB is the ion of choice because: 1) it yields a distinct dark blue color that is easily distinguishable from brown DAB; 2) the blue reaction product is very stable throughout the entire staining procedure; and 3) background staining is minimal. These findings can be applied to the double staining technique of two different antigens in the same section. Among three staining procedures discussed, the avidin-biotin peroxidase complex (Co-DAB)-peroxidase-antiperoxidase (DAB) technique produced the best results because: 1) no antibody elution was needed following the avidin-biotin-peroxidase complex procedure when the CoCl2-DAB modification was used; and 2) no background staining occurred.
发现三种金属离子，即氯化镍（NiCl2）、氯化钴（CoCl2）和硫酸铜（CuSO4），会改变通常呈棕色的二氨基联苯胺（DAB）反应的颜色。颜色范围从紫蓝色（NiCl2）、深蓝色/蓝黑色（CoCl2）到灰蓝色（CuSO4）。我们发现CoCl2 + DAB是首选离子，原因如下：1）它会产生明显的深蓝色，很容易与棕色的DAB区分开来；2）蓝色反应产物在整个染色过程中非常稳定；3）背景染色极少。这些发现可应用于同一切片中两种不同抗原的双重染色技术。在所讨论的三种染色方法中，抗生物素蛋白-生物素过氧化物酶复合物（Co-DAB）-过氧化物酶-抗过氧化物酶（DAB）技术产生的效果最佳，原因如下：1）当使用CoCl2-DAB修饰时，在抗生物素蛋白-生物素-过氧化物酶复合物步骤之后无需进行抗体洗脱；2）没有出现背景染色。
