Walking a thin line between fixation and epitope binding – characterization of antigen retrieval methods suitable for eosinophil and HSV-2 staining in formalin-fixed female reproductive tissue

Submitted: 29 November 2023
Accepted: 19 March 2024
Published: 16 April 2024
Abstract Views: 162
PDF: 127
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Antibody-based fluorescence analysis of female reproductive tissues in research of sexually transmitted diseases allows for an in-depth understanding of protein localization, interactions, and pathogenesis. However, in many cases, cryosectioning is not compatible with biosafety regulations; at all times, exposure of lab personnel and the public to potentially harmful pathogens from biological infectious material must be avoided; thus, formaldehyde fixation is essential. Due to formaldehyde’s cross-linking properties, protein detection with antibodies can be impeded. To allow effective epitope binding during immunofluorescence of formalin-fixed paraffin-embedded vaginal tissue, we investigated two antigen retrieval methods. We tested these methods regarding their suitability for automated image analysis, facilitating reproducible quantitative microscopic data acquisition in sexually transmitted disease research. Heat-based retrieval at 80°C in citrate buffer proved to increase antibody binding to eosinophil protein and HSV-2 visibly and tissue morphology best, and was the most efficient for sample processing and quantitative analysis.



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Ethics Approval

All protocols and experiments were approved by the Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Recklinghausen, Germany

Supporting Agencies

German Research Foundation (DFG) within the German-African Projects in Infectiology, German Center for Infection Research (DZIF), Bundesministerium fur Bildung und Forschung
William Horsnell, Wellcome Centre for Infectious Diseases Research in Africa, Institute of Infectious Disease and Molecular Medicine, Department of Pathology, Division of Immunology, University of Cape Town

Medical Research Council Centre for Medical Mycology, University of Exeter, UK

How to Cite

Wadephul, L. M., Arndts, K., Katawa, G., Dietlmeier, E., Horsnell, W., Hoerauf, A., & Ritter, M. (2024). Walking a thin line between fixation and epitope binding – characterization of antigen retrieval methods suitable for eosinophil and HSV-2 staining in formalin-fixed female reproductive tissue. European Journal of Histochemistry, 68(2). https://doi.org/10.4081/ejh.2024.3929