Double immunohistochemical staining in routine diagnostic pathology

Combining two different scientific disciplines – morphology and immunochemistry – immunohistochemistry has developed as an important instrument in research and clinical pathology. However, in some cases there is a need for knowledge about the relative localization of targets, which can only be obtained by vizualizing all relevant targets in one slide. Multiple staining can be defined as the detection of two or more targets on one slide. This technique enables the depiction of two or more targets/antigens in connection with other morphological features. We use the Benchmark ULTRA system in our lab, which is the next generation in IHC/ISH staining instrumentation. For the double staining method we use sequential staining. The most important step is to optimize the pretreatment step (time, buffer or protease) for both antigens. For the first detection step we selected ultraView Universal DAB system (brown) and for the second one ultraView Universal Alkaline Phosphatase Detection Kit (red).

The following combinations of antigens were determined as helpful: Ki 67 + CK 7; Ki 67 + CD 31; CD 34 + CK 7; CD 31 + p 53; D2- 40 + AE 1/3; CD 3 + CK 7; kappa + lambda light chains.

Conclusion: Detection of more targets/antigens in one slide has good results. Moreover, this method spares the tissue, which is very important in cases with small biopsy samples.