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Published on: September 3, 2017
An improved and cost-effective methodology for the reduction of autofluorescence in direct immunofluorescence studies
M S Viegas1, T C Martins, F Seco
1Molecular Pathology Laboratory, Portuguese Institute for Oncology of Coimbra (IPOCFG-EPE), Coimbra, Portugal. marta.s.viegas@gmail.com
Insights
Autofluorescence in confocal laser scanning microscopy (CLSM) hinders direct immunofluorescence. A novel method combining UV irradiation and Sudan Black B effectively reduces autofluorescence in diverse tissues, enabling clearer specific signal detection.
Area of Science:
- Immunohistochemistry
- Microscopy
- Biomedical Imaging
Background:
- Autofluorescence presents a significant challenge in immunofluorescence analysis using confocal laser scanning microscopy (CLSM).
- Minimizing tissue autofluorescence and background noise is crucial, especially for direct immunofluorescence studies.
- Existing methods for autofluorescence control are tissue-dependent and lack a universal application.
Purpose of the Study:
- To develop and validate a robust method for reducing autofluorescence in formalin-fixed, paraffin-embedded murine tissues.
- To improve upon existing techniques to allow for direct immunofluorescence labeling without compromising tissue integrity.
- To establish a generalizable approach for autofluorescence reduction applicable across different tissue types.
Main Methods:
- Evaluation of various autofluorescence reduction techniques on archival formalin-fixed murine liver, kidney, and pancreas sections.
- Application of a combined method involving short-duration, high-intensity UV irradiation and Sudan Black B staining.
- Assessment of the method's efficacy in reducing autofluorescence while preserving tissue morphology and direct immunofluorescence signal.
Main Results:
- Previously described autofluorescence reduction techniques were insufficient for direct immunofluorescence in the tested murine tissues.
- The combination of UV irradiation and Sudan Black B proved highly effective in reducing autofluorescence in both high-lipofuscin (liver, kidney) and low-lipofuscin (pancreas) tissues.
- This optimized methodology successfully lowered autofluorescent background to levels enabling the detection of specific signals via direct immunofluorescence.
Conclusions:
- A combined UV irradiation and Sudan Black B treatment offers a superior and broadly applicable solution for autofluorescence reduction in CLSM.
- This method overcomes limitations of previous techniques, enhancing the reliability of direct immunofluorescence in diverse biological samples.
- The validated approach facilitates clearer visualization and detection of specific targets in challenging tissues, improving immunofluorescence assay performance.
Abstract:
Interference by autofluorescence is one of the major shortcomes of immunofluorescence analysis by confocal laser scanning microscopy (CLSM). CLSM requires minimal tissue autofluorescence and reduced unspecific fluorescence background, requisites that become more critical when direct immunofluorescence studies are concerned. To control autofluorescence, different reagents and treatments can be used. Until now, the efficacy of the processes described depended on the tissue type and on the processing technique, no general recipe for the control of autofluorescence being available. Using paraffin sections of archival formalin-fixed murine liver, kidney and pancreas, we have found that previously described techniques were not able to reduce autofluorescence to levels that allowed direct immunofluorescence labelling. In this work, we aimed at improving currently described methodologies so that they would allow reduction of the autofluorescent background without affecting tissue integrity or direct immunofluorescence labelling. We have found that the combination of short-duration, high-intensity UV irradiation and Sudan Black B was the best approach to reduce autofluorescence in highly vascularised, high lipofuscins' content tissues, such as murine liver and kidney, and poorly vascularised, low lipofuscins' content tissues such as the pancreas. In addition, we herein show that this methodology is highly effective in reducing autofluorescent background to levels that allow detection of specific signals by direct immunofluorescence.
