DNA-barcode-based Multiplex Immunofluorescence Imaging to Analyze FFPE Specimens from Genetically Reprogrammed Murine Melanoma

1.6K views

09:58 min

June 6th, 2025

10.3791/68124-v

June 6th, 2025

1.6K views

Sachin S. Surwase^*¹^,²^,³ , Xin Ming M. Zhou^*³^,⁴ , Kathryn M. Luly¹^,²^,³ , Qingfeng Zhu⁵^,⁶^,⁷ , Niki Talebian⁶ , Robert A. Anders⁵^,⁶^,⁷^,⁸^,⁹ , Jordan J. Green¹^,²^,³^,⁵^,⁸^,⁹^,¹⁰^,¹¹^,¹² , Stephany Y. Tzeng¹^,²^,³ , Joel C. Sunshine¹^,³^,⁴^,⁵^,⁶^,⁸^,⁹

¹Department of Biomedical Engineering, Johns Hopkins University, ²Translational Tissue Engineering Center, Johns Hopkins University, ³Johns Hopkins Translational ImmunoEngineering Center, Johns Hopkins University, ⁴Department of Dermatology, Johns Hopkins University, ⁵Department of Oncology, Johns Hopkins University, ⁶Department of Pathology, Johns Hopkins University, ⁷Convergence Institute, Sidney Kimmel Comprehensive Cancer Center Johns Hopkins University School of Medicine, ⁸Bloomberg Kimmel Institute for Cancer Immunotherapy, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, ⁹Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, ¹⁰Institute for Nanobiotechnology, Johns Hopkins University, ¹¹Departments of Neurosurgery and Ophthalmology, Johns Hopkins University School of Medicine, ¹²Departments of Materials Science & Engineering and Chemical & Biomolecular Engineering, Johns Hopkins University

^* These authors contributed equally

This protocol provides a step-by-step guide for designing a multiplex immunofluorescence antibody panel for DNA-barcode-based imaging of murine FFPE melanoma tissues. We also describe an image analysis pipeline using open-source tools for generating spatial proteomics insights into the murine melanoma tumor immune microenvironment.

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Chapters in this video

0:00

Introduction

1:21

Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Staining and Imaging for Multiplex Analysis

4:37

Digital Pathology Analysis and Proteomics Data Normalization

8:34

Results

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