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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, Pennsylvania, United States of America.

Plos One
|March 5, 2026
PubMed

Insights

Immunohistochemistry (IHC) signal intensity can decrease over six weeks. Antibody choice and staining method significantly influence fluorescence stability, impacting data reliability.

Area of Science:

  • Biomedical Sciences
  • Microscopy Techniques
  • Immunohistochemistry

Background:

  • Immunohistochemistry (IHC) is a crucial technique in scientific research.
  • Signal intensity stability over time is an understudied aspect of IHC.
  • Changes in IHC signal intensity can affect data analysis and scientific conclusions.

Purpose of the Study:

  • To investigate the stability of IHC signal intensity over a six-week period.
  • To determine factors influencing fluorescence intensity changes in IHC staining.
  • To provide recommendations for reliable IHC imaging and data collection.

Main Methods:

  • Assessed fluorescence intensity stability over six weeks using widefield and confocal microscopy.
  • Evaluated the impact of primary and secondary antibodies on signal stability.
  • Compared chemical staining versus IHC staining methods.

Main Results:

  • Fluorescence intensity can decrease significantly over a six-week period.
  • The extent of signal decrease is influenced by antibody selection and combinations.
  • Differences in stability were observed between chemical and IHC staining methods.

Conclusions:

  • IHC signal intensity is not always stable over time.
  • Careful selection of antibodies and staining protocols is essential for reproducible IHC results.
  • Best practices for imaging are reinforced to ensure accurate data for cell counting, protein expression, and colocalization studies.

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