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Longitudinal Assessment of Fluorescence Stability Shows Fluorescence Intensity Decreases Over Time: Implications for
Sean C Sweat1,2,3, Sarah P R Berg1, Tenzin Kunkhyen1
1Department of Neurobiology, University of Pittsburgh, Pittsburgh PA 15213.
Insights
Immunohistochemistry (IHC) signal intensity can decrease over six weeks. Antibody choice and staining method impact fluorescence stability, affecting data reliability.
Area of Science:
- Biomedical Sciences
- Microscopy Techniques
- Immunohistochemistry
Background:
- Immunohistochemistry (IHC) is a crucial technique in biological and clinical research.
- Reliable IHC staining requires careful optimization, but signal stability over time is understudied.
- Signal intensity changes can significantly impact data interpretation and scientific conclusions.
Purpose of the Study:
- To investigate the stability of IHC fluorescence signal intensity over a six-week period.
- To determine factors influencing signal decay in IHC staining.
- To provide recommendations for best practices in imaging fluorescently labeled samples.
Main Methods:
- Assessed fluorescence intensity stability over six weeks using widefield and confocal microscopy.
- Evaluated the influence of primary antibody, secondary antibody, and antibody combinations.
- Compared chemical staining with IHC staining methods.
Main Results:
- Fluorescence intensity significantly decreased over the six-week assessment period.
- Signal decay was influenced by the specific primary and secondary antibodies used.
- The choice of antibody combination and staining method (chemical vs. IHC) affected the extent of signal loss.
Conclusions:
- IHC signal intensity is not permanently stable and can decrease over time.
- Careful selection of antibodies and consistent imaging protocols are essential for reliable IHC data.
- Adhering to best practices in imaging fluorescent staining ensures accurate quantification and interpretation.
Abstract:
Immunohistochemistry (IHC) is one of the most widely used techniques across basic, translational, and clinical sciences. Key considerations need to be made to achieve reliable and robust IHC staining, however what has been understudied is the stability of IHC signal intensity over time. Changes in signal intensity over time have significant implications for data analysis and interpretation and ultimately impact scientific conclusions. In order to explore changes in IHC signal, the stability of fluorescence intensity was assessed over the course of six weeks using widefield or confocal microscopy. Results indicate that fluorescence intensity can decrease over this time course and that whether this decrease occurs and to what extent is influenced by the selection of the primary antibody as well as that of the secondary antibody, primary-secondary antibody combination, and utilization of chemical staining versus IHC staining. This investigation reinforces best practices for imaging fluorescent staining to ensure accurate and reliable data collection, be it for cell counting, assessing protein expression levels, or marker colocalization.
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