Immunofluorescence Microscopy
Immunofluorescence Microscopy - Related Videos
01:12
Immunofluorescence Microscopy
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2023
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. The...
09:56
Immunofluorescence Microscopy: Immunofluorescence Staining of Paraffin-Embedded Tissue Sections
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2023
Source: Thomas Chaffee1, Thomas S. Griffith2,3,4, and Kathryn L. Schwertfeger1,3,4 1 Department of Lab Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455 2 Department of Urology, University of Minnesota, Minneapolis, MN 55455 3 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455 4 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 Pathologic analyses of tissue sections can be used to obtain a better understanding of normal tissue...
05:23
Immunofluorescence Microscopy for the Analysis of DNA Double-Strand Breaks
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2025
In this video, we describe the immunofluorescence microscopy technique to detect specific DNA damage response proteins localized at the sites of DNA double-strand breaks in human mononuclear cells. The proteins are recognized by specific primary antibodies, which in turn bind to fluorophore-tagged secondary antibodies that fluoresce during microscopic visualization, enabling visualization of the proteins as distinct fluorescent foci within the cell nuclei.
09:00
Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy
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Cited by 21 •
2014
The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.
16:31
Simple Elimination of Background Fluorescence in Formalin-Fixed Human Brain Tissue for Immunofluorescence Microscopy
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Cited by 43 •
2017
Background autofluorescence of biological samples often complicates fluorescence-based imaging techniques, especially in aged human postmitotic tissues. This protocol describes how autofluorescence from these samples can be effectively removed using a commercially available light emitting diode light source to photobleach the sample prior to immunostaining.
FAQs
Indirect immunofluorescence microscopy can amplify the detectable signal because a fluorescent secondary antibody is used after an unlabeled primary antibody has recognized the target. Direct immunofluorescence attaches the fluorophore to the antibody that binds the target itself. This distinction affects how the labeling arrangement is organized when researchers choose between direct detection and signal amplification.
The fluorophore produces detectable fluorescence when the specimen is illuminated at a wavelength appropriate for that label. This illumination step converts antibody-associated labeling into an observable signal, allowing the target’s location to be mapped within cells or tissues. Consequently, the optical condition is central to visualizing molecular distribution rather than merely identifying antibody binding.
Using multiple fluorophores allows researchers to examine several targets in the same specimen while retaining their shared spatial context. Each labeled target contributes to the fluorescence image, so the resulting comparison can show where molecules occur relative to one another. This supports studies of cellular organization and interactions between proteins without separating the targets into entirely different specimens.
Preserving spatial context connects molecular detection with the architecture of the cell or tissue in which the target occurs. Instead of reporting protein expression without location, the image can reveal molecular localization alongside cell structure. That relationship is especially useful when biology depends on where a molecule is found, including developmental processes, disease mechanisms, and cellular organization.
A basic workflow proceeds from target recognition to optical detection. An antibody system is placed in contact with the cells or tissue, using either a fluorophore-linked binding antibody or an unlabeled primary antibody followed by a fluorescent secondary antibody. Appropriate illumination then makes the labeled target detectable, allowing its distribution to be examined in the original specimen.
Researchers choose Immunofluorescence Microscopy when they need to connect a molecule’s presence with its location in a biological specimen. It is suited to investigations of cellular organization, developmental processes, disease mechanisms, and protein interactions. The method is therefore valuable when spatial information adds meaning to changes in protein expression or to the arrangement of structures within cells and tissues.
By examining fluorescence associated with a target across cells or tissues, researchers can study where protein expression changes occur while retaining the specimen’s structure. The method therefore links expression patterns to localization, rather than treating expression as an isolated measurement. This perspective can support analyses of disease mechanisms, developmental processes, and cellular organization in their spatial biological context.