Multiplex chromogenic immunohistochemistry to stain and analyze paraffin tissue sections from the mouse or human

Here we describe a multiplex chromogenic immunohistochemistry platform to stain and analyze two markers in paraffin tissue sections from mouse or human. The basis of the protocol is a series of stripping and re-probing steps with subsequent image analysis, which allows the user to perform multiplex imaging in a reliable and affordable manner. Here, we describe specific usage to assess the levels of PD-L1 in tumor-associated macrophages. We have used different antibodies and assessed this protocol for up to five consecutive antibodies per slide. For complete details on the use and execution of this protocol, please refer to Orgaz et al. (2020).¹.