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Related Experiment Videos

Improved double immunofluorescence for confocal laser scanning microscopy.

R K Kumar1, C C Chapple, N Hunter

  • 1School of Pathology, University of New South Wales, Sydney, Australia.

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society
|August 17, 1999
PubMed
Summary

New Alexa fluorochromes (Alexa 488 and Alexa 568) enable reliable double immunofluorescence labeling for confocal microscopy. These dyes provide excellent signal separation and co-localization, overcoming limitations of single laser systems.

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Area of Science:

  • Immunology
  • Microscopy
  • Biochemistry

Background:

  • Effective double immunofluorescence labeling in confocal laser scanning microscopy depends on distinct fluorochrome signal separation.
  • Limitations exist in achieving optimal excitation of dyes with well-separated emission spectra using a single argon ion laser.

Purpose of the Study:

  • To introduce and evaluate novel sulfonated rhodamine fluorochromes, Alexa 488 and Alexa 568, for improved double immunofluorescence labeling.
  • To demonstrate the effectiveness of these fluorochromes in achieving reliable co-localization signals in confocal microscopy.

Main Methods:

  • Utilized Alexa 488 (green-emitting) and Alexa 568 (red-emitting) fluorochromes for double labeling.
  • Employed signal amplification for the more abundant antigen using a biotinylated bridging antibody and labeled streptavidin.

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  • Incorporated appropriate controls to verify the absence of crosstalk between fluorescence channels.

    • Main Results:

    • Alexa 488 provided brighter images compared to fluorescein, with similar photodegradation.
    • Alexa 568, combined with signal amplification, effectively visualized the abundant antigen.
    • The combination of Alexa 488 and Alexa 568 allowed for unequivocal demonstration of co-localization.
    • Successful signal separation was achieved, overcoming single argon ion laser limitations.

    Conclusions:

    • The novel Alexa 488 and Alexa 568 fluorochromes offer a reliable method for double immunofluorescence labeling in confocal microscopy.
    • This fluorochrome combination facilitates clear co-localization analysis and may benefit users with alternative laser systems.