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J Immunol. 2019 Jan 1;202(1):292-299. doi: 10.4049/jimmunol.1800878. Epub 2018 Dec 3.

Improved Multiplex Immunohistochemistry for Immune Microenvironment Evaluation of Mouse Formalin-Fixed, Paraffin-Embedded Tissues.

Author information

1
Division of Surgical Oncology, Department of Surgery, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX 75390.
2
Centre for Cancer Immunology, Faculty of Medicine, Southampton General Hospital, University of Southampton, Southampton 016 6YD, United Kingdom.
3
Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030; and.
4
Division of Surgical Oncology, Department of Surgery, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX 75390; rolf.brekken@utsouthwestern.edu.
5
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390.

Abstract

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 formalin-fixed, 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 effective Ag-retrieval buffer. We also validated 22 Abs specific for mouse immune cell markers to distinguish B cells, T cells, NK cells, 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 and Ab panels in the quantitation and spatial distribution of immune cells.

PMID:
30510069
PMCID:
PMC6310091
[Available on 2020-01-01]
DOI:
10.4049/jimmunol.1800878

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