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Related Experiment Videos

Immunostaining for cell picking and real-time mRNA quantitation.

L Fink1, T Kinfe, W Seeger

  • 1Department of Pathology, Justus-Liebig-University, Giessen, Germany. ludger.fink@patho.med.uni-giessen.de

The American Journal of Pathology
|November 14, 2000
PubMed
Summary

Combining laser-assisted cell picking with immunofluorescence and mRNA quantitation allows accurate analysis of specific cell types within complex tissues. This method reliably measures changes in gene expression, such as in stimulated alveolar macrophages.

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Area of Science:

  • Molecular Biology
  • Immunohistochemistry
  • Cell Biology

Background:

  • Microdissection enables cell-specific mRNA analysis in complex tissues.
  • Real-time RT-PCR allows mRNA quantitation from small cell samples.
  • Cell-type specific immunostaining is crucial for precise cell identification.

Purpose of the Study:

  • To evaluate the effect of immunofluorescence staining on mRNA quantitation.
  • To assess the reliability of combining laser-assisted cell picking with mRNA analysis.
  • To quantify mRNA levels in specific rat alveolar macrophages (AMs) after stimulation.

Main Methods:

  • Alveolar macrophages (AMs) were analyzed from control and stimulated rat lungs.
  • Immunofluorescence staining identified AMs in tissue sections.

Related Experiment Videos

  • Laser-assisted sampling picked 15 AM profiles per sample.
  • Real-time RT-PCR quantified nitric oxide synthase II (NOSII) and tumor necrosis factor-alpha (TNF-alpha) mRNA.

    • Main Results:

    • Stimulated lungs showed a significant increase in NOSII and TNF-alpha mRNA-positive AMs.
    • A marked increase in target gene mRNA to standard gene mRNA ratio was observed in stimulated AMs.
    • Results from laser-assisted sampling closely matched direct RNA extraction from lavaged AMs.

    Conclusions:

    • Optimized immunofluorescence protocols are compatible with laser-assisted cell picking and mRNA quantitation.
    • This combined approach reliably analyzes gene expression in immunohistochemically characterized cells within complex tissues.
    • The technique accurately quantifies mRNA changes in specific cell populations, like stimulated alveolar macrophages.