The Third Affiliated Hospital of Kunming Medical University and Yunnan Cancer Hospital 1 article published in JoVE Cancer Research Multiplex Cyclic Fluorescent Immunohistochemistry Yun Chen*1, Hui Zhang*2, Yaodong Fan3, Liu Yang4,5, Yan Dong*1 1Department of Pathology, The Third Affiliated Hospital of Kunming Medical University & Yunnan Cancer Hospital, 2Department of Gastroenterology, Wu Han NO.1 Hospital, 3Department of Neurosurgery, The Third Affiliated Hospital of Kunming Medical University & Yunnan Cancer Hospital, 4Departments of Biomedical Research Center, The Affiliated Calmette Hospital of Kunming Medical University & The First Hospital of Kunming, 5School of Medical, Yunnan University Multiplex cyclic immunohistochemistry allows in situ detection of multiple markers simultaneously using repeated antigen-antibody incubation, image scanning, and image alignment and integration. Here, we present the operating protocol for identifying immune cell substrates with this technology in lung cancer and paired brain metastasis samples.