Cyclic Multiplex Fluorescent Immunohistochemistry Protocol to Phenotype Glial Cells in Formalin-Fixed Paraffin-Embedded Human Brain Sections

Abstract

There is a growing interest in expanding the multiplexing capability of immunohistochemistry to achieve a deeper phenotyping of various cell types in health and disease. Here, we describe a protocol of cyclic multiplex fluorescent immunohistochemistry that enables the labeling of up to 16 antigens on the same formalin-fixed paraffin-embedded section using "off-the-shelf," commercially available, primary antibodies as well as fluorescently conjugated secondary antibodies. Key steps include the denaturing/stripping of the antibodies by microwaving and the quenching of any remaining fluorescent signal between the cycles of otherwise traditional multiplexed fluorescent immunohistochemistry. We have successfully applied this protocol to characterize astrocytic and microglial responses to Aβ plaques and neurofibrillary tangles in Alzheimer's disease brains, but it could be easily adapted to other user's needs regarding cell types, disease, and organ.

Keywords: Alignment; Amyloid-β plaques; Antibodies; Astrocytes; Cyclic multiplex fluorescence immunohistochemistry; Formalin-fixed paraffin-embedded sections; Glial cells; Microglia; Neurofibrillary tangles; Segmentation.