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Custom Stellaris® FISH Probes
Product Description
Spheroplasting buffer (10 mL Buffer B + 100 µL of 200 mM vanadyl ribonucleoside complex) A set of Stellaris FISH Probes comprises up to 48 single-labeled oligonucleotides designed to selectively bind to targeted transcripts. Stellaris FISH Probes bound to target RNA produce fluorescent signals to permit detection of single RNA molecules as diffraction-limited spots by conventional WARNING! Formamide is a teratogen that is easily
absorbed through the skin and should be used in a
Storage Guidelines
Stellaris FISH Probes are shipped dry and can be stored frozen WARNING! Be sure to let the formamide warm to room
temperature before opening the bottle.
Final composition is 100 mg/mL dextran sulfate and Short-Term Storage & Daily Use Dissolved probe mix should be subjected to a minimum number of freeze-thaw cycles. For daily and short-term use, the mix can be stored at +2 to +8 °C in the dark for 1 mL 20X saline-sodium citrate (SSC), nuclease-free We recommend freezing probes in the dark at -15 to Nuclease-free water to 10 mL final volume -30 °C for storage lasting longer than a month.
WARNING! Formamide is a teratogen that is easily
Reconstituting the Dried Probe Stock
absorbed through the skin and should be used in a The probe stock should be sufficient to provide up to 200 or more hybridizations. For frequent use, aliquots of the probe WARNING! Be sure to let the formamide warm to room
stock can be stored at +2 to +8 °C in the dark for up to a Redissolve the dried oligonucleotide probe blend in 200 µL of Final composition is 10% formamide in 2X SSC T E buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), to create a probe stock at a total oligo concentration of 25 µM. Mix well Reagents and Equipment
Nuclease-free water to 50 mL final volume Fixation buffer (50 mL) for all sample types except Nuclear Stain for use after hybridization: 4’,6-diamidino-2-phenylindole (DAPI) dissolved in wash Final composition is 3.7% formaldehyde in 1X PBS. Mix:
5 mL 37% Formaldehyde solution
5 mL 10X Phosphate Buffered Saline (PBS), RNase free
Nuclease-free water to 50 mL final volume
Buffer B (1.2 M sorbitol, 0.1 M potassium phosphate, pH 7.5) Vanadyl Ribonucleoside Complex (200 mM) Anti-fade buffer and enzymes (GLOX buffer):
Note: The GLOX buffer is recommended for use with
Standard fluorescence microscope (e.g., Nikon TE2000 probes labeled with Quasar 570 and Quasar 670 dyes, and is incompatible with fluorescein dye. For probes Strong light source, such as a mercury or metal-halide labeled with fluorescein, instead use commercially lamp (xenon or LED are typically not bright enough).
available antifades such as Vectashield Mounting Filter sets appropriate for the fluorophores.
Medium (Vector Labs, catalog #H1000).
Standard cooled CCD camera, ideally optimized for Final composition of the GLOX buffer without low-light level imaging rather than speed (13 µm pixel enzymes is 0.4% glucose in 10 mM Tris, 2X SSC Technical Support
40 µL 10% glucose in water10 µL 1 M Tris-HCl, pH 8.0 If you require additional information or technical assistance please feel free to e-mail our Technical Support Group at: techsupport@biosearchtech.com. Our knowledgeable staff Vortex and then transfer 100 µL of this GLOX buffer to is also available for telephone consultation from 8:00 AM another tube, to which add 1 µL of the glucose oxidase to 5:00 PM, Monday through Friday, Pacific Time. Please stock (glucose oxidase diluted to 3.7 mg/mL in 50 mM sodium acetate, pH ~5, catalog #G0543 from Sigma or equivalent) and 1 µL of mildly vortexed catalase 1.800.GENOME.1 (436.6631) US & Canada only suspension (catalog #C3155 from Sigma or equivalent). The remainder can be used as an equilibration buffer.
Note: The GLOX buffer should not be allowed to dry
or cure, but rather image the sample immediately after References
mounting.
Alternatively, for convenience, commercially available 1. Raj A, Tyagi S. Detection of individual endogenous premixed anti-fade solutions such as Cytoseal (Richard- RNA transcripts in situ using multiple singly labeled Allan or Amazon) or Prolong Gold (Life Technologies) probes. Methods in Enzymology 2010; 472: 365-86. can be used, but have been shown to be less effective 2. Femino, A. M., Fay, F. S., Fogarty, K., and Singer, R. H. than the GLOX buffer, and their use may result in Visualization of single RNA transcripts in situ. Science Lab-Tek chambered coverglass, catalog #43300-772
from VWR or equivalent in conjunction with #1 Square
coverslips, catalog # 48366-045 from VWR or equivalent.
Note: Chambered coverglass is only compatible with
an inverted scope. Instead use standard slides and
coverslips for an upright scope.
#1 Round coverslips, catalog # 48380-046 from VWR
or equivalent, in conjunction with cell culture plates
from catalog # EK-680111 E&K Scientific, for use on an
upright scope.
81 Digital Drive, Novato, CA 94949-5728 USA | info@biosearchtech.com 2012 Biosearch Technologies, Inc. All rights reserved.
This product is sold under license from the University of Medicine and Dentistry of New Jersey and may be used under its patent rights for Research Use Only. Information on licensing programs for the commercial and/or diagnostic use of these products is available by contacting licensing@biosearchtech.com. “Stellaris” is a registered trademark of Biosearch Technologies, Inc., “Biosearch Technologies, Inc.” is a trademark of Biosearch Technologies, Inc., “Advancing Nucleic Acid Technology” is a servicemark of Biosearch Technologies, Inc.
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