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Fluorescence fading and stabilization in cytofluorometry.

M Fukuda, Y Tsuchihashi, T Takamatsu

    Histochemistry
    |January 1, 1980
    PubMed
    Summary
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    This study investigated factors influencing fluorescence fading in cytofluorometry. Mounting specimens in non-fluorescent resin with methanol fixation significantly stabilized fluorescence, preventing fading even after two years.

    Area of Science:

    • Cell Biology
    • Biophysics
    • Analytical Chemistry

    Background:

    • Fluorescence fading is a significant challenge in cytofluorometry, impacting data reliability.
    • Understanding factors affecting fluorescence stability is crucial for accurate cellular analysis.

    Purpose of the Study:

    • To investigate factors influencing fluorescence fading in nuclear staining and cytofluorometry.
    • To identify methods for enhancing fluorescence stability in stained specimens.

    Main Methods:

    • Examined effects of RNase, trypsin, and hypotonic solutions on fluorescence fading.
    • Tested various nuclear stains (pararosaniline Feulgen, Hoechst 33258, acriflavine-SO2, cresyl-violet-SO2) with pure DNA.
    • Evaluated different mounting media (glycerin, buffer, Entellan) and post-staining fixation (methanol).

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    Main Results:

    • Specimen treatments like RNase and trypsin accelerated fluorescence fading.
    • Glycerin and buffer solutions resulted in rapid fluorescence decay for tested fluorochromes.
    • Mounting in Entellan resin with methanol fixation markedly stabilized fluorescence for all tested fluorochromes, including fluorescein isothiocyanate (FITC).
    • No fading was observed for indirect immunofluorescence with anti-UV-DNA antibody after 2 years of storage.

    Conclusions:

    • Factors promoting conformational stability of macromolecule-dye complexes enhance fluorescence stabilization.
    • Post-staining fixation with methanol and mounting in non-fluorescent resin are effective strategies to prevent fluorescence fading in cytofluorometry.