Multiplex fluorescence in situ hybridization (FISH) enables you to assay multiple targets and visualize colocalized signals in a single specimen. Using spectrally distinct fluorophore labels for each hybridization probe, this approach gives you the power to resolve several genetic elements or multiple gene expression patterns through multicolor visual display.

Life Technologies offers FISH Tag™ detection kits for routine analysis, and TSA™ kits for very rare or low-abundance targets.


FISH Tag™ detection kits

FISH Tag™ detection kits provide the labeling reagents and buffers you need to generate optimal FISH probes for multiplex assays. In a simple protocol, nick translation (for DNA probes) or in vitro transcription (for RNA probes) is used to enzymatically incorporate amine-modified nucleotides, followed by chemical labeling with amine-reactive Alexa Fluor® dyes.

Compared to dye-labeled nucleotides, aminoallyl-modified nucleotides are consistently incorporated at high levels and covalently labeled using reliable succinimidyl ester coupling chemistry. The end result is a higher degree of labeling and improved signal-to-noise ratios in FISH applications.

FISH Tag™ kits offer a complete workflow solution for FISH applications and deliver exceptional signal intensity and photostability. With a direct imaging protocol and spectrally distinct dyes, you can view multiple targets simultaneously.

  FISH Tag™ detection kits
Multiplex FISH detection using FISH Tag™ RNA and TSA kits combined in a whole mount Drosophila embryo.
The expression of Krupple (magenta) and rhomboid (orange) were detected using FISH Tag™ RNA Kits with Alexa Fluor® 647 dye and Alexa Fluor® 555 dye or the Multicolor Kit. The expression of dpp was performed with a fluorescein-labeled RNA probe (ChromaTide® fluorescein-12-UTP, and detected with TSA™ Kit #2 with Alexa Fluor® 488 tyramide and anti-fluorescein/Oregon Green®, rabbit IgG fraction, horseradish peroxidase conjugate.

TSA™ detection kits

For rare or very low-abundance targets, the fluorescence signals of FISH probes can be amplified using tyramide-based signal amplification (TSA™ technology). TSA™ kits employ horseradish peroxidase (HRP) to generate high-density fluorescence labeling at the site of probe hybridization. The technique is ideal for low-abundance DNA and RNA targets and is compatible with multiplex strategies.

The most common TSA™ technology approach is to bind a haptenylated probe to the specimen, followed by secondary detection of the probe with an HRP-labeled antibody or streptavidin conjugate.

Soluble dye-labeled tyramides are activated by HRP and are covalently deposited at the site of probe hybridization, providing excellent resolution and high signal-to-noise ratios.

Life Technologies provides Alexa Fluor® dye tyramides as well as those conjugated to Oregon Green® dye and biotin.

  Detection of the Golgi apparatus using Tyramide Signal AmplificationDetection of the Golgi apparatus using tyramide signal amplification (TSA™ technology).
Formaldehyde-fixed and Triton® X-100–permeabilized BPAE cells were blocked with goat serum, nuclei were stained with DAPI (blue), and F-actin was stained with Alexa Fluor® 488 phalloidin (green). Anti–golgin-97 IgG1 was labeled using TSA™ Kit #5 with HRP–goat anti–mouse IgG and Alexa Fluor® 594 tyramide to indicate cell-to-cell adhesion (red).

TSA™ detection kits for low-abundance targets

Labeled tyramide
Horseradish peroxidase conjugate

Anti–mouse IgG (host: goat)

Anti–rabbit IgG (host: goat)
Streptavidin
Alexa Fluor® 350 T20917 T20927 T20937
Alexa Fluor® 488 T20912 T20922 T20932
Oregon Green® 488 T20939
Alexa Fluor® 546 T20913 T20923 T20933
Alexa Fluor® 555 T30953 T30954 T30955
Alexa Fluor® 568 T20914 T20924 T20934
Alexa Fluor® 594 T20915 T20925 T20935
Alexa Fluor® 647 T20916 T20926 T20936
Biotin-XX T20911 T20921 T20931

Sample protocols

Fluorescence in situ hybridization (FISH)Enlarge Image
  Fluorescence in situ hybridization (FISH)Enlarge Image
Four-color fluorescence in situ hybridization on a Drosophila embryo. A late blastoderm stage (nuclear cycle 14) embryo was probed with four different RNA probes.
Blue: sog labeled with DNP, followed by a rabbit anti–dinitrophenyl-KLH IgG antibody detected with an Alexa Fluor® 647 chicken anti–rabbit IgG antibody. Green: ind labeled with biotin, followed by streptavidin HRP and Alexa Fluor® 350 tyramide (TSA Kit #27. Red: msh labeled with digoxigenin followed by sheep anti-digoxigenin antibody detected with an Alexa Fluor® 488 donkey anti–sheep IgG antibody. Yellow: sna labeled with fluorescein followed by mouse anti-fluorescein antibody detected with an Alexa Fluor® 555 goat anti–mouse IgG antibody. Image contributed by Dave Kosman and Ethan Bier, University of California, San Diego.
  Simultaneous detection of expression of five genes in a whole-mount Drosophila embryo by fluorescence in situ hybridization (FISH) with five RNA probes.
Red: sog labeled using aminoallyl UTP and Alexa Fluor® 647 succinimidyl ester. Green: ind labeled with DNP, followed by rabbit anti–dinitrophenyl-KLH IgG antibody prelabeled with the Zenon® Alexa Fluor® 555 Rabbit IgG Labeling Kit. Blue: en labeled with biotin and detected with HRP–streptavidin and Alexa Fluor® 405 tyramide (TSA™ Kit #39, T30952). Yellow: wg labeled with digoxigenin and detected with sheep anti–digoxigenin IgG antibody and Alexa Fluor® 594 Donkey Anti–Sheep IgG antibody. Magenta: msh labeled with fluorescein and detected with mouse anti–fluorescein/Oregon Green® IgG2a antibody and Alexa Fluor® 488 Goat Anti–Mouse IgG antibody. Image contributed by Dave Kosman and Ethan Bier, University of California, San Diego.
     
Fluorescence in situ hybridization (FISH)Enlarge Image   Fluorescence in situ hybridization (FISH)Enlarge Image
In situ hybridization of a-satellite probes to human chromosomes 1, 15 and 17 detected by tyramide signal amplification.
a-Satellite probes to chromosomes 1, 15 and 17 were labeled by nick translation with biotin-11-dUTP, ChromaTide® Texas Red®-12-dUTP and ChromaTide® Oregon Green® 488-5-dUTP, respectively. Following simultaneous hybridization of all three probes, the biotinylated chromosome 1 probe was detected with HRP–streptavidin conjugate and Alexa Fluor® 546 tyramide (TSA Kit #23). HRP activity from this first TSA detection step was then quenched by treatment with 1% hydrogen peroxide for 30 minutes. Lastly, the Oregon Green® 488 dye–labeled chromosome 17 probe was detected with anti-fluorescein/Oregon Green® antibody followed by HRP-conjugated goat anti–mouse IgG antibody and Alexa Fluor® 594 tyramide (TSA Kit #5). HRP activity from this second TSA detection step was then quenched by treatment with 1% hydrogen peroxide for 30 minutes. The Texas Red® dye–labeled chromosome 15 probe was then detected with rabbit anti–Texas Red® antibody followed by HRP-conjugated goat anti–rabbit IgG antibody and Alexa Fluor® 488 tyramide (TSA Kit #12. After counterstaining with Hoechst 33258, the images were acquired using filters appropriate for DAPI, FITC, TRITC and the Texas Red® dye.
  Fluorescence in situ hybridization detected by tyramide signal amplification. Chromosome spreads were prepared from the cultured fibroblast cell line MRC-5 and hybridized with a biotinylated a-satellite probe specific for chromosome 17.
The probe was generated by nick translation in the presence of ChromaTide® biotin-11-dUTP. For detection by TSA, hybridized chromosome spreads were labeled using TSA Kit #22 with HRP–streptavidin and Alexa Fluor® 488 tyramide (upper panel) or with TSA Kit #23 with HRP–streptavidin and Alexa Fluor® 546 tyramide (lower panel). After counterstaining with DAPI, images were obtained using filters appropriate for DAPI, FITC or TRITC.

Resources

Molecular Probes® Handbook
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