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Related Concept Videos

Immunofluorescence Microscopy01:12

Immunofluorescence Microscopy

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A fluorescence microscope uses fluorescent chromophores called fluorochromes, which can absorb energy from a light source and then emit this energy as visible light. Fluorochromes include naturally fluorescent substances (such as chlorophylls) and fluorescent stains that are added to the specimen to create contrast. Dyes such as Texas red and FITC are examples of fluorochromes. Other examples include the nucleic acid dyes 4’,6’-diamidino-2-phenylindole (DAPI), and acridine orange.
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Immunocytochemistry (ICC) and immunohistochemistry (IHC) are techniques that use antibodies to check for specific proteins or antigens in a sample. The technique was first published by Albert Coons in 1941 to detect the presence of pneumococcal antigen in tissue sections from mice infected with Pneumococcus. Immunocytochemistry helps localization of proteins or antigens in individual cells like blood cells, stem cells, etc., while immunohistochemistry does the same for tissue samples.
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DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
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Related Experiment Video

Updated: Oct 11, 2025

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons
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Double Labeling Fluorescent Immunocytochemistry.

Monika Rak1,2, Krzysztof Reiss3

  • 1Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Kraków, Kraków, Poland. monika.rak@uj.edu.pl.

Methods in Molecular Biology (Clifton, N.J.)
|December 3, 2021
PubMed
Summary

This study details a fluorescent immunocytochemistry protocol for double labeling LC3 and IRS-1 proteins in human glioblastoma cells. The method enhances understanding of cellular components and their functions through precise antigen detection.

Keywords:
AntibodyCellsImmunocytochemistryImmunofluorescenceLocalization

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Area of Science:

  • Cell Biology
  • Immunocytochemistry
  • Microscopy

Background:

  • Fluorescent immunocytochemistry is crucial for analyzing cell composition, structure, and function.
  • The technique relies on advancements in antibody production, fluorescent labels, and microscopy.
  • Effective implementation requires knowledge of procedure planning and execution for reliable results.

Purpose of the Study:

  • To describe a detailed protocol for the double labeling of LC3 and IRS-1 proteins in LN-229 human glioblastoma cells.
  • To highlight critical procedural steps and potential variables in fluorescent immunocytochemistry.
  • To provide a reliable method for investigating protein colocalization in glioblastoma.

Main Methods:

  • Protocol development for double labeling specific proteins (LC3 and IRS-1) in glioblastoma cell lines.
  • Application of fluorescently conjugated antibodies for antigen detection.
  • Utilizing advanced microscopy techniques for visualizing protein localization and colocalization.

Main Results:

  • Successful double labeling of LC3 and IRS-1 proteins was achieved in LN-229 cells.
  • Identification of key procedural steps influencing the reliability of results.
  • Demonstration of protein colocalization patterns within glioblastoma cells.

Conclusions:

  • The described protocol provides a robust method for fluorescent immunocytochemistry in glioblastoma research.
  • Understanding procedural variables is essential for accurate antigen detection and colocalization analysis.
  • This technique advances the study of cellular components and their functional interactions in cancer biology.