The PhenoCycler System utilizes reagents and buffers that have been optimized to be used in combination with PhenoCycler antibodies, barcodes and reporters. For each imaging cycle, three spectrally distinct PhenoCycler Reporters, are applied to the stained tissue to visualize the corresponding PhenoCycler antibody. A series of washes remove the PhenoCycler Reporters, followed by the application of the next set of reporters.