Improved multiplex immunohistochemistry for immune microenvironment evaluation of mouse formalin-fixed, paraffin-embedded tissues

Immune profiling of tissue through multiplex immunohistochemistry is important for the investigation of immune cell dynamics, and it can contribute to disease prognosis and evaluation of treatment response in cancer patients. However, protocols for mouse formalinfixed, paraffin-embedded tissue have been less successful. Given that formalin fixation and paraffin embedding remains the most common preparation method for processing mouse tissue, this has limited the options to study the immune system and the impact of novel therapeutics in preclinical models. In an attempt to address this, we developed an improved immunohistochemistry protocol with a more effectiveAg-retrieval buffer.We also validated 22 Abs specific formouse immune cellmarkers to distinguish B cells,T cells, NKcells, macrophages, dendritic cells, and neutrophils. In addition, we designed and tested novel strategies to identify immune cells for which unique Abs are currently not available. Last, in the 4T1 model of breast cancer, we demonstrate the utility of our protocol andAb panels in the quantitation and spatial distribution of immune cells.