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Published on: July 11, 2025
Characterizing and Diminishing Autofluorescence in Formalin-fixed Paraffin-embedded Human Respiratory Tissue
A Sally Davis1, Anke Richter1, Steven Becker1
1Viral Pathogenesis and Evolution Section (ASD, JEM, AS, JKT), Division of Intramural Research, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MarylandBioimaging Section, Research Technology Branch (SB), Division of Intramural Research, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MarylandLaboratory of Immunogenetics (JS), Division of Intramural Research, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MarylandDefense Resources Management Institute, School of International Graduate Studies, Naval Postgraduate School, Monterey, California (AR)Department of Population Health and Pathobiology, North Carolina State University College of Veterinary Medicine, Raleigh, North Carolina (ASD).
Abstract:
Tissue autofluorescence frequently hampers visualization of immunofluorescent markers in formalin-fixed paraffin-embedded respiratory tissues. We assessed nine treatments reported to have efficacy in reducing autofluorescence in other tissue types. The three most efficacious were Eriochrome black T, Sudan black B and sodium borohydride, as measured using white light laser confocal Λ2 (multi-lambda) analysis. We also assessed the impact of steam antigen retrieval and serum application on human tracheal tissue autofluorescence. Functionally fitting this Λ2 data to 2-dimensional Gaussian surfaces revealed that steam antigen retrieval and serum application contribute minimally to autofluorescence and that the three treatments are disparately efficacious. Together, these studies provide a set of guidelines for diminishing autofluorescence in formalin-fixed paraffin-embedded human respiratory tissue. Additionally, these characterization techniques are transferable to similar questions in other tissue types, as demonstrated on frozen human liver tissue and paraffin-embedded mouse lung tissue fixed in different fixatives.
