Related Experiment Video
Updated: May 1, 2026
Optimized Workflow for Iterative Bleaching Extends Multiplexity Imaging of Highly Autofluorescent Clinical Samples
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).
Reducing autofluorescence in fixed respiratory tissues is crucial for immunofluorescence. Eriochrome black T, Sudan black B, and sodium borohydride were most effective, with minimal impact from antigen retrieval or serum application.
Area of Science:
- Histology and Pathology
- Biomedical Imaging
- Fluorescence Microscopy
Background:
- Tissue autofluorescence in formalin-fixed paraffin-embedded (FFPE) respiratory tissues significantly obstructs immunofluorescent marker visualization.
- Existing methods for autofluorescence reduction often lack specific validation for respiratory tissues.
Purpose of the Study:
- To evaluate and compare the efficacy of nine different treatments for reducing autofluorescence in FFPE human respiratory tissues.
- To assess the contribution of steam antigen retrieval and serum application to autofluorescence.
- To establish guidelines for minimizing autofluorescence in FFPE respiratory tissues for improved immunofluorescence.
Main Methods:
- Screening of nine candidate autofluorescence-reducing treatments on FFPE human respiratory tissue.
- Quantitative analysis of autofluorescence using white light laser confocal multi-lambda (Λ²) analysis.
- Modeling of Λ² data using 2-dimensional Gaussian surfaces to assess treatment efficacy and contributing factors.
Main Results:
- Eriochrome black T, Sudan black B, and sodium borohydride demonstrated the highest efficacy in reducing autofluorescence.
- Steam antigen retrieval and serum application were found to contribute minimally to the overall autofluorescence.
- Significant differences in efficacy were observed among the tested treatments.
Conclusions:
- Specific chemical treatments, notably Eriochrome black T, Sudan black B, and sodium borohydride, are highly effective for reducing autofluorescence in FFPE respiratory tissues.
- The study provides practical guidelines for optimizing immunofluorescence imaging in FFPE respiratory samples.
- The characterization techniques are adaptable for autofluorescence assessment in other tissue types and fixation methods.
