Colocalization Detection
Colocalization Detection - Related Videos
03:13
Immunostaining to Visualize Viral Antigen and Host Protein Colocalization in the Insect Gut
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2026
Source: Zhang, L., et al. Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts. J. Vis. Exp. (2021)The video demonstrates the preparation of gut tissue from a plant virus–infected insect vector for fluorescence imaging. Fixed guts were washed, permeabilized, and incubated with two fluorescent antibodies in a blocking solution—one targeting a viral antigen in vesicles, the other a vesicle-associated membrane protein. The tissue was counterstained for actin...
09:10
Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce
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Cited by 17 •
2010
This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).
07:13
Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry
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Cited by 8 •
2016
Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.
07:42
Super-Resolution Imaging and Shared Management: A Protocol for Confocal Microscopy with Multiplex Detection
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2026
This protocol details the advanced imaging applications, and shared-resource management of a laser-scanning confocal microscope system integrated with a multiplexed array detector for high-resolution cellular and subcellular imaging. The workflow enables multimodal imaging from confocal to super-resolution (~120 nm) within a single platform, making nanoscale visualization more accessible.
09:00
Detection of Protein Ubiquitination
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Cited by 96 •
2009
Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.
FAQs
Accurate channel alignment ensures that corresponding pixels or objects represent the same physical location in the specimen. Misalignment can create apparent separation or overlap that does not reflect the underlying biology. In neuroscience images, this step is especially important when comparing labeled proteins, receptors, or neuronal compartments across separately acquired fluorescence channels.
Pearson’s correlation evaluates how changes in signal intensity correspond between two channels, whereas Manders’ overlap coefficients quantify the proportion of one signal occupying locations marked by the other. These measures therefore describe different aspects of association. Reporting the appropriate metric helps distinguish intensity correlation from the extent of spatial signal overlap.
Background correction removes signal that is unrelated to the labeled structures, while thresholding determines which intensities count as meaningful signal. Both choices can substantially influence calculated association or overlap. Applying them carefully helps prevent weak background fluorescence from being interpreted as colocalization and makes comparisons between neuronal samples more reliable.
A typical workflow begins by acquiring separate fluorescence channels, aligning them, and correcting background. The analyst then applies suitable thresholds and compares the channels pixel by pixel or object by object. Measures such as Pearson’s correlation or Manders’ overlap coefficients can then summarize the spatial relationship for the selected cellular region.
This approach is useful when researchers need to examine whether labeled components occupy shared regions within neurons. Applications described for neuroscience include evaluating protein distribution, examining neurotransmitter receptors at synapses, and mapping molecular components in axons or dendrites. The selected cellular compartment provides the spatial context for interpreting the measured association.
The analysis can reveal spatial coincidence between signals and help compare the distribution of molecular components across neuronal structures. However, overlapping fluorescence does not by itself demonstrate direct molecular interaction. Researchers therefore need careful controls and should interpret the measured association as evidence of shared location rather than definitive physical binding or interaction.