invitrogen 00-8401 Mode D'emploi

Cat. No. 00-8401
®
SPOT-Light Tissue Pretreatment kit
For Enzyme and Heat Pretreatment of FFPE Tissue prior to CISH
For use in heat pretreatment and enzyme digestion of Formalin-Fixed, Paraffin-Embedded (FFPE) tissue prior to Chromogenic In Situ Hybridization
(CISH) detection.
INTENDED USE
For In Vitro Diagnostic Use. CAUTION: Not for human or animal therapeutic use. Uses other than the labeled intended use may be a violation of
local law.
REAGENTS PROVIDED
1. One Liter of Heat Pretreatment Solution, pH 7.0 (Ready to Use)
2.
One 10 ml bottle of Enzyme Reagent (Ready To Use)
STORAGE
Store at 2-8°C.
STABILITY
There are no obvious signs to indicate instability of these reagents. They have been quality controlled to assure consistent and reliable performance.
Do not use after the expiration date stamped on container. With proper storage there is no significant loss of performance. If the reagents are stored
under any conditions other than those specified, those conditions must be validated by the user.
TISSUE PREPARATION
Slides should be treated with an adhesive before mounting tissue section. Deparaffinize slides and dehydrate tissue sections.
Perform heat pretreatment and enzyme digestion as recommended in the instructions that come with the ISH probe. The following procedure may be
used if probe instructions are unavailable.
HEAT PRETREATMENT
®
1. Heat 500 mL SPOT -Light
15 minutes (Note: different incubation times may be required depending on tissue fixation. A 15 minute incubation is a recommended starting
point).
2. Wash in PBS or dH 0 at room temperature (RT) for 2 x 3 minutes.
2
ENZYME DIGESTION
1. Cover tissue with 100-200 µL of Enzyme Reagent (Reagent 2) for 10 min at RT.
2. Note: depending on tissue fixative used, different incubation times may be required. Excessive digestion will cause loss of nuclei and chromosome
structure. Please refer to Troubleshooting section for details.
3. Wash in PBS or dH 0 at RT for 3 x 2 minutes.
2
4. Dehydrate slides in a series of 70%, 85%, 95%, and 100% ethanol for 2 minutes each at room temperature, air dry, and proceed to denaturation
and hybridization.
TROUBLESHOOTING
1. Throughout the entire procedure, unless otherwise indicated, it is important that the tissue section does not dehydrate.
2. Heat Pretreatment (The most critical step for successful CISH performance): The specimen must be boiled or heated above 98°C for 15 min.
in Heat Pretreatment Solution.
3. Enzyme Digestion (A critical step for successful CISH performance): Different enzyme incubation times (5-15 min.) may be required,
depending on tissue type and fixation method. For most breast tissues, 10 min. enzyme digestion at room temperature (RT) will produce the
best CISH results. Be sure to pre-warm the Enzyme Pretreatment Reagent to RT prior to adding to the tissue section. Enzyme
pretreatment of the specimen should be evaluated immediately at the completion of the CISH protocol. If nuclei are not counterstained and there is
an absent or very weak CISH signal, this may be due to nuclear loss as the result of excessive digestion. If nuclei are strongly counterstained but a
CISH signal is absent in the nuclei, this may be due to under-digestion during the pepsin pretreatment. As an alternative, enzyme pretreatment may
also be performed at 37˚C for 3 minutes if optimal results are not attained.
4. Probe denaturation at a temperature lower than recommended by the protocol may result in a weak or absent CISH signal.
5. Hybridization performed for shorter time periods, or stringent washes performed at higher temperatures, than recommended by the protocol may
produce a decrease in or complete loss of the CISH signal.
6.
Please contact your local distributor or Invitrogen at
product literature.
General ISH REFERENCES
1. Wilkinson, D.G., In Situ Hybridization, a Practical Approach. 2
2. Polak, J.M. and McGee, J. In Situ Hybridization: Principles and Practice. Oxford University Press, Oxford, UK, (1998).
3. Verma, R.S and Babu, A. Human Chromosomes: Principles and Techniques. 2
4. Leitch, A.R. et al. In Situ Hybridization-A Practical Guide: Royal Microscopy Society Microscopy Handbooks. Vol 27, Bios Scientific Publishers, Oxford, UK,
(1994).
TRADEMARK
® ®
Zymed and SPO T -Light are trademarks of Invitrogen Corporation.
Authorized Representative for IVDD 98/79/EC: Invitrogen Ltd., Inchinnan Business Park, 3 Fountain Drive, Paisley, PA4 9RF, UK
PI008401
© 2007 Invitrogen Corporation. All right reserved. These products may be covered by one or more Limited Use Label Licenses (see the Invitrogen Catalog or www.invitrogen.com).
By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses.
Tissue Pretreatment Solution (Reagent 1) in a beaker on a hot plate until it is either boiling or 98-100°C. Boil slides for
tech_support@invitrogen.com
nd
Invitrogen Corporation • 542 Flynn Rd • Camarillo • CA 93012 • Tel: 800.955.6288 • E-mail:
or 1-800-955-6288 for additional technical assistance or
ed., Oxford university Press, Oxford, (1998).
nd
ed., Health Professions Division, New York, (1995).
www.invitrogen.com
techsupport@invitrogen.com
(Rev 12/09) DCC-09-1898