Multiplex Cyclic Fluorescent Immunohistochemistry

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.

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and spatially organizing immune cell subsets and target proteins are crucial for prognostic and therapeutic purposes. This has led to the development of multiplexed immunohistochemistry staining methods. Multiplex fluorescence immunohistochemistry allows the simultaneous detection of multiple markers, facilitating a comprehensive understanding of cell function and intercellular interactions. In this paper, we describe a workflow for the multiplex cyclic fluorescent immunohistochemistry assay and its application in the quantification analysis of lymphocyte subsets. The multiplex cyclic fluorescent immunohistochemistry staining follows similar steps and reagents as standard immunohistochemistry, involving antigen retrieval, cyclic antibody incubation, and staining on a formalin-fixed paraffin-embedded (FFPE) tissue slide. During the antigen-antibody reaction, a mixture of antibodies from different species is prepared. Conditions, such as antigen retrieval time and antibody concentration, are optimized and validated to increase the signal-to-noise ratio. This technique is reproducible and serves as a valuable tool for immunotherapy research and clinical applications.

Brain metastases (BM) represent the most common central nervous system (CNS) tumors, occurring in nearly half of non-small cell lung cancer cases (NSCLC), with a poor prognosis¹. An estimated 10%-20% of NSCLC patients already have BM at the time of initial diagnosis, and approximately 40% of NSCLC cases will develop BM during the course of treatment². The tumor microenvironment (TME) is closely associated with NSCLC occurrence and BM, including various components, such as blood vessels, fibroblasts, macrophages, extracellular matrix (ECM), lymphoid, bone marrow-derived immune cells, and signaling molecules

The research was approved by the medical ethics committee of Yunnan Cancer Hospital/the Third Affiliated Hospital of Kunming Medical University. All the subjects/legal guardians signed informed consent.

1. Slide preparation

1. Cut sections of paired paraffin blocks containing primary lung tumor or lung cancer brain metastases cells at a thickness of 4 µm using a microtome. Remove sections to water and separate with tweezers, choose the best one and adhere it onto.......

We present a protocol for cyclic antigen detection using 5-color multiplex fluorescence on a single slide. Through our optimization of the assay, we enable the incubation of two antibodies from different species (Figure 1). The necessary devices for the experiment procedure include a pressure cooker and immunostaining box (Figure 2A).

After completing the assay, we define pseudo color of the four markers before scanning the slides. Th.......

We have described the process for multiplex cyclic fluorescence immunohistochemistry staining. The primary antibody selection is a crucial aspect of the fluorescence immunohistochemistry assay, and monoclonal antibodies are recommended for better specificity and repeatability. To optimize the working concentration of the primary antibody, a series of dilutions have been tested through immunohistochemistry experiments. Both positive controls (to assess target antigen expression) and negative controls (no primary antibody .......

This work was supported by the National Natural Science Foundation of China (NO.81860413, 81960455), Yunnan Science and Technology Department Fund (202001AY070001-080), Scientific Research Foundation of Education Department of Yunnan Province(2019J1274).