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Updated: May 22, 2026
DNA-barcode-based Multiplex Immunofluorescence Imaging to Analyze FFPE Specimens from Genetically Reprogrammed Murine Melanoma
Published on: June 6, 2025
Simultaneous immunofluorescent labeling using anti-BrdU monoclonal antibody and a melanocyte-specific marker in
Morea Petersen1, Lester M Davids, Susan H Kidson
1Faculty of Health Sciences, University of Cape Town, South Africa.
Insights
This study developed a new method for simultaneously labeling proliferating cells and melanocytes in human skin samples. This robust protocol enhances accuracy for studying cell populations in fixed tissues.
Area of Science:
- Histology
- Immunohistochemistry
- Dermatology
Background:
- Accurate immunolabeling is crucial for consistent biological data.
- Multiple immunolabeling is essential for identifying cell phenotypes and locations within the same compartment.
- Standard 5-bromodeoxyuridine (BrdU) immunolabeling methods have limitations for multiple antigen detection.
Purpose of the Study:
- To develop a robust protocol for simultaneous BrdU and melanocyte-specific antigen labeling in formalin-fixed paraffin-embedded (FFPE) human skin.
- To overcome technical limitations of standard methods for multiple immunolabeling.
- To enable accurate assessment of proliferating melanocytes in FFPE skin samples.
Main Methods:
- Human skin samples were obtained and incubated with BrdU.
- Standard FFPE tissue processing was employed.
- Heat-induced antigen retrieval was performed, followed by double immunolabeling with anti-BrdU and anti-Melan A/MART-1 antibodies, using fluorescent secondary antibodies for detection.
Main Results:
- Successful simultaneous detection of proliferating cells (BrdU-positive) and melanocytes (Melan A/MART-1-positive) was achieved.
- Both cell types were localized to the basal compartment of the epidermis.
- The optimized protocol demonstrated the feasibility of double labeling in FFPE human skin.
Conclusions:
- Heat-induced epitope retrieval is critical for successful double labeling, replacing harsh pretreatment methods.
- A robust protocol for double labeling of proliferating cells and melanocytes/melanoblasts in FFPE human skin has been optimized.
- This method facilitates the study of cell proliferation and melanocyte dynamics in fixed skin tissues.
Abstract:
Immunolabeling of tissue sections requires careful optimization of protocols in order to achieve accurate and consistent data. Multiple immunolabeling is desirable when determining the exact location and phenotype of cell populations in the same cellular compartment. 5-bromodeoxyuridine (BrdU)-immunolabeling is commonly used to assess cellular proliferation in vitro. However, the technical limitations of standard methods preclude multiple antigen immunolabeling. The aim was therefore to develop a robust protocol for simultaneous labeling using anti-BrdU and a melanocyte-specific marker in formalin-fixed paraffin-embedded (FFPE) skin samples. Human skin samples were obtained from patients undergoing elective plastic surgery. The tissue was incubated with BrdU, and a standard sample procedure for FFPE tissue was used. Heat-induced antigen retrieval was performed in a conventional pressure cooker, followed by immunolabeling with anti-BrdU and anti-Melan A/MART-1 antibodies. Fluorescent-conjugated secondary antibodies were used for signal detection. We have demonstrated both proliferating cells (BrdU-immunopositive) and melanocytes (Melan A/MART-1-immunopositive) in the basal compartment of the epidermis in our skin samples. Successful double labeling requires heat-induced epitope retrieval to replace the harsh pretreatment protocols of standard BrdU immunolabeling methods. We have optimized a robust protocol for the double labeling of proliferating cells and cells bearing melanocyte-specific antigens (melanocytes and/or melanoblasts) in FFPE human skin samples.
