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Cytolocalization artifacts with immunofluorescent probes.

S J Friedman, C L Dewar, P Skehan

    Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire
    |December 1, 1986
    PubMed
    Summary
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    Common cell preparation methods like formaldehyde fixation and detergent extraction can create artifacts, altering lectin binding and localization results in microscopy studies. Researchers must be cautious about these artifacts when interpreting cytolocalization data.

    Area of Science:

    • Cell Biology
    • Microscopy Techniques
    • Biochemistry

    Background:

    • Formaldehyde fixation, nonionic detergent extraction, and ligand binding are standard methods for visualizing cellular components using immunofluorescence microscopy.
    • These techniques are frequently employed in cytolocalization studies to examine antigen and lectin-reactive molecule distribution within cytoskeletal preparations.

    Purpose of the Study:

    • To investigate the potential for artifacts introduced by formaldehyde fixation and nonionic detergent extraction during cytolocalization studies.
    • To assess the impact of these common procedures on wheat germ agglutinin (WGA) binding and localization in BeWo choriocarcinoma cells.

    Main Methods:

    • Wheat germ agglutinin (WGA) binding assays using radiolabeled (125I-WGA) and fluorescently labeled (FITC-WGA) lectins.

    Related Experiment Videos

  • Analysis of WGA binding patterns and localization in intact BeWo cells before and after formaldehyde fixation.
  • Evaluation of the effects of nonionic detergent extraction on WGA binding activity in both lectin-pretreated and non-pretreated cells.
  • Preparation of nuclear-cytoskeletal monolayers for binding activity assessment.

    • Main Results:

    • Formaldehyde fixation reduced 125I-WGA binding by 30% and altered FITC-WGA staining patterns, decreasing surface binding except at perinuclear sites.
    • Nonionic detergent extraction solubilized 50% of bound lectin in pretreated cells, while binding sites remained active in the detergent-insoluble fraction.
    • Nuclear-cytoskeletal monolayers from non-lectin-pretreated cells showed significant loss of WGA binding activity.

    Conclusions:

    • Formaldehyde fixation and nonionic detergent extraction can introduce significant artifacts into cytolocalization studies.
    • These artifacts can lead to erroneous conclusions regarding the association of cytoskeletal components with membrane molecules when using immunofluorescence microscopy on fixed and extracted cells.
    • Careful consideration of fixation and extraction protocols is crucial for accurate interpretation of lectin binding and cytolocalization data.