Immunofluorescence Quantification
Immunofluorescence Quantification - Related Videos
08:15
Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries
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Cited by 8 •
2018
Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.
06:25
Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR
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Cited by 5 •
2020
We describe an immunofluorescence and quantitative PCR method for the localization and quantification of begomoviruses in insect tissues. The immunofluorescence protocol can be used to colocalize viral and vector proteins. The quantitative PCR protocol can be extended to quantify viruses in whole whitefly bodies and virus-infected plants.
09:12
High-Throughput Automated Multiplex Immunofluorescence Assays for Translational Research
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2025
This report describes an automated protocol for multiplex immunofluorescence (mIF) assays on an automated slide stainer. It demonstrates the high reproducibility of spatial proteomics on standard high-throughput tissue autostainers and whole-slide fluorescence imagers, which is ideal for custom mIF assays across a large number of tissue slides in translational research.
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...
05:34
Dual Immunofluorescence of γH2AX and 53BP1 in Human Peripheral Lymphocytes
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Cited by 6 •
2023
This protocol presents a method to assess the formation and repair of DNA double-strand breaks through the simultaneous detection of γH2AX and 53BP1 foci in interphase nuclei of bleomycin-treated human peripheral lymphocytes.
FAQs
The primary antibody recognizes the selected antigen, while the fluorescent secondary antibody provides the signal that can be detected in an image. This two-antibody arrangement connects molecular recognition with measurable fluorescence. Because the measured signal depends on both labeling and imaging, consistent antibody use and controlled acquisition are important when comparing molecule abundance across cells, tissue regions, or experimental groups.
Background fluorescence can make a target appear more abundant than it is, while differences in image acquisition can alter measured intensity independently of biology. Quantification therefore requires accounting for background and controlling imaging conditions. Normalization further improves comparisons by reducing the influence of technical variation, allowing observed differences to be interpreted more confidently between brain regions or experimental groups.
The analysis can quantify fluorescence intensity, labeled area, or the number of cells meeting the measurement criteria. Intensity provides information about signal abundance, area describes the extent of labeled structures, and cell counts estimate how many cells display the selected marker. Choosing among these readouts depends on whether the study emphasizes molecular level, spatial extent, or cellular prevalence.
Spatial measurements show where a molecule or structure occurs within cells or tissue rather than only indicating how much signal is present overall. This distinction can reveal changes in cellular organization and help relate molecular patterns to disease mechanisms. In brain studies, regional comparisons are especially relevant when neuronal, glial, synaptic, or pathological markers may vary across tissue locations.
A typical workflow pairs an antigen-specific primary antibody with a fluorescent secondary antibody, captures images under controlled conditions, and measures the resulting signal. Analysis may include intensity, labeled area, or cell counts, followed by correction for background and normalization of imaging-related variation. Applying the same workflow across samples supports quantitative comparisons among regions and experimental groups.
Immunofluorescence quantification is useful when researchers need quantitative evidence about neuronal markers, glial proteins, synaptic components, or pathological inclusions. It can compare these signals across brain regions or experimental groups and support studies of neurodevelopment, neurodegeneration, and treatment response. The resulting measurements can connect molecular changes with cellular organization and help evaluate differences associated with disease or intervention.