Stellaris® FISH Probes, Custom Assay with FAM (Fluorescein, Emission 520 nm)
Custom Stellaris® RNA FISH Probe Set labeled with FAM (Fluorescein) dye to detect your RNA of interest.
Stellaris RNA FISH is a RNA visualization method that allows simultaneous detection, localization, and quantification of individual RNA molecules at the cellular level in fixed samples using fluorescence microscopy.
A set of Stellaris RNA FISH Probes is comprised of a pool of up to 48 unique probes, each labelled with a fluorophore, that collectively bind along an RNA target transcript to produce a diffraction limited spot.
Before considering designing a Stellaris RNA FISH Customized Probe Set, please also check out our Stellaris RNA FISH DesignReady Probe Sets for a large selection of professionally designed targets.
We also recommend selecting a Stellaris RNA FISH Positive Control (ShipReady) Probe Set RNA to complement your experiment.
Stellaris Buffers are the perfect accompaniment to your experiment, allowing for even more robust RNA detection and analysis through reduced background signal.
FAM (Fluorescein) Dye
The Fluorescein modification incorporates a fluorescein (FAM) moiety at the terminus of an oligonucleotide. FAM fluoresces in the green region of the visible spectrum.
Special Notes and Considerations: The green channel where FAM fluoresces is not ideal for RNA FISH as there tends to be high auto-fluorescence from the specimen itself. This auto-fluorescence can make it difficult to discern true RNA transcripts from background, and is especially true in tissue where auto-fluorescence is generally already higher. Because of this, probe labeled with FAM perform best in cultured cells. Additionally we recommend the use of FAM-labeled probe sets with >35 individual probes, and as a compliment to a primary probe set. Usage should be limited to specimens with known low autofluorescence, fixed with MeOH/HAC, and when other filter options are not available.
Anti-fade Recommendations: The FAM dye requires an anti-fade mounting media and is compatible with Vectashield by Vector Labs (H-1000) and Prolong Diamond by ThermoFisher (P36961). Please note that FAM is incompatible with enzymatic glucose-oxidase/catalase (GLOX) antifade.
How long will my sample last after mounting?: The FAM signal should be imaged immediately. Hardmount Prolong Diamond will allow for spot verification up to 5 days, but with greatly reduced signal to noise ratio after the first day.
Filter Recommendation: Our in-house team uses Chroma's 49011-ET-FITC/Alexa Fluor 488/Fluo3/Oregon Green single band filter set.
- Name: Fluorescein
- Formula: C24H18NO8
- Absorption maximum (lambda max): 495 nm
- Extinction coefficient at lambda max: 71,300 M-1cm-1
- Extinction coefficient at 260 nm: 26,900 M-1cm-1
- Fluorescence maximum: 520 nm
Please read the tech article titled, “Designing Stellaris RNA FISH Probes” before designing your custom assay.
You can also use Chroma’s Spectra Viewer to check your microscope’s dye filter set compatibility. Use the Stellaris Dyes and Modifications page to select the right dye for your microscope and multiplexing recommendations.
Note I: Due to the variable autofluorescence in the green channel inherent to some cells and tissues, it is not recommended to use FAM for some Stellaris RNA FISH assays. We recommend testing your sample for green channel autofluorescence before ordering FAM labeled sets or ordering an alternative dye that is compatible with your experiment.
Note II: The success rate of Custom Probe Sets depend on many variables, such as imaging platform, operator use of optics, target sequence, transcript number, subcellular localization of transcript, number of probes in the set, space between probes, masking level, sample identity whether cultured cells or fresh or frozen tissue, preparation and handling of sample, and customer's design of probe sets with or without the Stellaris Probe Designer. The customer is responsible for designing a probe set that is compatible with all of the variables mentioned above unique to their platform technologies and model system, and therefore we cannot guarantee the success of Customer Probe Sets, or troubleshoot the designs.
Please contact us by email, phone or using this form for further advise and details.
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