Multiplex Fluorescent Hybridization
Multiplex Fluorescent Hybridization - Related Videos
10:15
Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)
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Cited by 21 •
2011
An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.
13:14
Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications
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Cited by 4 •
2015
Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.
02:36
Multiplexed Fluorescence Imaging of Viral Nucleic Acids and Protein in Infected Human Lymphocytes
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2026
Source: Shah, R., et al. Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection. J. Vis. Exp. (2020)This video demonstrates simultaneous visualization of HIV-1 DNA, RNA, and capsid protein in infected human lymphocytes using branched DNA hybridization and immunofluorescence, enabling detailed analysis of viral replication stages within individual cells.
07:36
Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections
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Cited by 3 •
2021
This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.
09:00
Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes
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Cited by 45 •
2012
Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.
FAQs
Denaturation separates the DNA strands and temporarily removes the existing base-pairing structure. This allows each labeled probe to reanneal specifically to its complementary target sequence rather than remaining associated with the original DNA partner. The resulting selective binding is the basis for assigning fluorescence signals to particular genomic sequences and supports accurate visualization of multiple targets in the same specimen.
Each probe carries a fluorescent label that produces a distinguishable signal when it binds its matching DNA sequence. Because the targets receive different fluorophores, fluorescence microscopy can separate their signals and show their locations simultaneously. This color-based distinction allows several chromosome regions or sequences to be evaluated in one assay instead of interpreting a single target in isolation.
Multiplexing combines several probe-target measurements within one hybridization assay. That design improves efficiency while preserving information about multiple genomic locations, enabling a more comprehensive view of chromosome and genome organization. Examining the signals together can also reveal relationships among targets that would be less apparent if each sequence were assessed in a separate experiment.
The workflow begins with fluorescently labeled probes and DNA containing the sequences of interest. Both the target DNA and probes are denatured, after which the probes reanneal to complementary sequences. The prepared material is then examined by fluorescence microscopy, where the resulting signals are used to locate and compare the selected genetic targets.
In genetics, the method supports chromosome identification and gene mapping, allowing researchers to relate fluorescent signals to particular chromosomal or genomic locations. It can also reveal copy-number changes and structural abnormalities. These capabilities make the assay useful when the goal is to examine several aspects of genome organization within the same cellular or chromosomal preparation.
The combined pattern of distinct fluorescent signals provides positional information for multiple DNA sequences at once. Comparing where those signals appear on cells or chromosomes can expose altered relationships among genomic regions, including structural abnormalities and complex rearrangements. This multi-target view helps investigators interpret genome organization beyond what a single fluorescent probe could show.