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.

We offer Invitrogen™ FISH Tag™ detection kits for routine analysis, and Invitrogen™ SuperBoost™ 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 Invitrogen™ 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 tyramide signal amplification combined in a whole mount Drosophila embryo.
The expression of Krüppel (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 an anti-fluorescein/Oregon Green dye antibody (rabbit IgG fraction, horseradish peroxidase conjugate) and Alexa Fluor 499 tyramide reagent.

SuperBoost Signal Amplification Kits

With sensitivity 10 to 200 times that of standard ICC/IHC/ISH methods, SuperBoost kits offer superior signal amplification and definition and clarity for high-resolution imaging of low-abundance targets. Combining the brightness of Alexa Fluor dyes with trusted poly-HRP mediated tyramide signal amplification, SuperBoost generates sensitivity 2 to 10 times above standard solutions like Invitrogen™ TSA™, or TSA™ PLUS reagents. For imaging that demands definition and clarity, bring your targets out of the background with SuperBoost signal amplification kits.

SuperBoost signal amplification detection kits

Labeled tyramide (Ex/Em)
Tyramide SuperBoost Kits*
Anti–mouse IgG (host: goat)
Anti–rabbit IgG (host: goat)
Streptavidin
Alexa Fluor 488 (495/519 nm) B40912
B40941 (50 coverslips)
B40922
B40943 (50 coverslips)
B40932
Alexa Fluor 555 (555/565 nm) B40913 B40923 B40933
Alexa Fluor 594 (591/617 nm) B40915
B40942 (50 coverslips)
B40925
B40944 (50 coverslips)
B40935
Alexa Fluor 647 (650/668 nm) B40916 B40926 B40936
Biotin-XX B40911 B40921 B40931
* Unless otherwise stated, sufficient material is provided for up to 150 18 mm x 18 mm coverslips (if using 150 µL in most critical incubation steps). Volumes can be adjusted for samples of different sizes.

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 Invitrogen™ Alexa Fluor™ 350 tyramide. 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 Invitrogen™ Alexa Fluor™ 405 tyramide). 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.
α-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. HRP activity from this first TSA detection step was then quenched by treatment with 1% hydrogen peroxide for 30 minutes. Lastly, the Invitrogen™ 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 Invitrogen™ Alexa Fluor™ 594 tyramide. HRP activity from this second TSA detection step was then quenched by treatment with 1% hydrogen peroxide for 30 minutes. The Invitrogen™ 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 Invitrogen™ Alexa Fluor™ 488 tyramide. 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 with HRP–streptavidin and Alexa Fluor 488 tyramide (left panel) or with HRP–streptavidin and Invitrogen™ Alexa Fluor™ 546 tyramide (right panel). After counterstaining with DAPI, images were obtained using filters appropriate for DAPI, FITC or TRITC.

Resources

Molecular Probes Handbook
Related technologies