A novel and practical method using HRP-polymer conjugate and microwave treatment for visualization of 2 antigens raised from the same or different species in paraffin-embedded tissues.

Recent advances in imaging systems have improved the ability to simultaneously visualize expression of multiple antigens in a single tissue section. In most instances, fluorescence microscopy, modern imaging systems, electronic scanners, and expensive reagents are required to achieve these results. Using conventional methodologies, we have developed an inexpensive and straightforward method for visualizing 2 antigens on a single formalin-fixed paraffin-embedded tissue section. The protocol combines the use of broad spectrum horseradish peroxidase-polymer conjugate and microwave treatment for detection of 2 primary antibodies raised from the same or different species. Our technique enables the researcher to simultaneously visualize 2 antigens in paraffin-embedded tissue requiring only one enzyme and a microwave.