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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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Immunoelectron microscopy utilizes immunogold labeling of endogenous proteins with specific antibodies to detect and localize these proteins in cells and tissues. The procedure provides insights into the distribution and quantification of protein under different stimulation conditions offering clues about their functions. Conjugating highly electron-dense gold particles with primary or secondary antibodies allow antigen detection on and within cells, with high resolution and specificity.
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Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.
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Super-resolution Fluorescence Microscopy01:37

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Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...
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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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Updated: Jul 20, 2025

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus
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Immunofluorescence Microscopy.

Domenico F Galati1, David J Asai2

  • 1Biology Department, College of Science and Engineering, Western Washington University, Bellingham, Washington.

Current Protocols
|August 4, 2023
PubMed
Summary
This summary is machine-generated.

Immunofluorescence microscopy visualizes cellular molecules using fluorescent antibodies. This guide details protocols for various cell types and advanced imaging techniques, including super-resolution microscopy.

Keywords:
Tetrahymenacytoskeletonfluorescenceimmunofluorescencemicrotubulestubulin antibody

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Area of Science:

  • Cell Biology
  • Microscopy
  • Molecular Biology

Background:

  • Visualizing fluorescence-tagged molecules reveals cellular dynamics.
  • Immunofluorescence microscopy uses fluorescently labeled antibodies to detect specific molecules.
  • Effective application requires careful consideration of antigen, antibody, fixation, and imaging.

Purpose of the Study:

  • To provide detailed protocols for immunofluorescence staining and imaging.
  • To cover diverse cell types, including adherent fibroblasts and suspension Tetrahymena.
  • To enable visualization using widefield, confocal, and super-resolution microscopy.

Main Methods:

  • Protocol for immunofluorescence staining of adherent cells (fibroblasts).
  • Protocol for immunofluorescence staining of suspension cells (Tetrahymena).
  • Protocols for visualization using widefield, laser scanning confocal, and super-resolution (SRRF) microscopy.

Main Results:

  • Demonstrated protocols for successful immunofluorescence staining in fibroblasts and Tetrahymena.
  • Enabled visualization of cytoskeleton and organelles in different cell types.
  • Facilitated imaging across a range of fluorescence microscopy techniques.

Conclusions:

  • Immunofluorescence microscopy is a versatile technique for visualizing cellular structures.
  • Standardized protocols can be adapted for various cell types and advanced imaging.
  • This work provides a comprehensive resource for researchers using immunofluorescence microscopy.