Набор реагентов для пробоподготовки POSEIDON FISH Digestion Kit
POSEIDON FISH Digestion Kit
KBI-60006 FISH Digestion Kit Older or difficult samples or slides with cytoplasmic background Version:IfU KBI-60006 FISH digestion kit_D1.0 Published: June 2009 Page 1 of 2 Instructions for use for KBI-60006 FISH Digestion Kit in combination with POSEIDON™ Repeat-Free™ fluorescent labeled DNA probes
Fluorescent in situ hybridization (FISH) identifies or labels target genomic sequences so that their location can be studied. DNA sequences from appropriate chromosome specific probes are first labeled with reporter molecules. The labeled DNA probe is then hybridized to the metaphase chromosomes or interphase nuclei on a slide. After washing, the specimen is screened for the reporter molecules by fluorescence microscopy. POSEIDONTM Repeat-FreeTM probes do not contain Cot-1 DNA. Hybridization efficiency is therefore increased and background, due to unspecific binding, is highly reduced. For use on metaphase and interphase cells from peripheral blood cultures or direct preparations prepared by standard cytogenetic methods see: The ACT cytogenetics laboratory manual. 2nd ed. New York: Raven Press; 1991. This pretreatment kit is specifically developed to obtain optimal results in case older samples, slides with cytoplasmic background, or difficult samples are used or expected. Note: freshly prepared cytological samples It is advised to use KBI-60005 FISH reagent kit in case freshly prepared cytological samples are used. For paraffin embedded tissues it is recommended to use pretreatment kits KBI-60004 or KBI-60007. For more info consult our website: www.kreatech.com Pretreatment: 1. Pretreat dry sample slide in 2 x SSC (LK-104B), at 37°C for 2 minutes (min). 2. Drop approximately 200 µl of Pepsin Solution (LK- 101B) on the cells and incubate at room temperature the slides 30 seconds - 10 min (depending on sample material, fixation time and storage conditions). 3. Wash slide for 3 min in 1 x PBS at room temperature. 4. Post-fix in 1% buffered formaldehyde in 1x PBS / 20 mM MgCl2 for 10 min at room temperature. 5. Wash slide for 3 min in 1 x PBS at room temperature.
6. Dehydrate in 70%, 85% and 100% ethanol for 1 min each. Air-dry at room temperature. 7. Proceed with Probe preparation. Note: Check protein digestion and pretreatment by applying 15 µl DAPI counterstain and evaluate slides using a fluorescence microscope equipped with a DAPI filter. Remove cover slip and soak tissue in 2 x SSC for 2 min and prolong protein digestion if sample is not sufficiently digested. Use a fresh sample and reduce protein digestion time if the sample is overdigested. Probe preparation: ON, PN, and MD POSEIDON Repeat-Free probes are supplied Ready to Use (RtU). SE, ST, and WC POSEIDON Repeat-Free probes are provided 5 x concentrated and must be diluted as follows: 2 µl 5 x conc. Probe in 8 µl FISH Hybridization Buffer (FHB or WCB, supplied with probes). To combine several 5 x conc. probes, replace FISH Hybridization Buffer (FHB or WCB) by 2 µl for each probe added. Co-denaturation: Apply 10 µl of probe or probe-mix per 22 x 22 mm field. Cover with glass cover slip and seal with Fixogum or rubber cement. Denature sample and probe on a hot plate at 75 ±1 °C for 5-10 min. Continue with hybridization. Note: Probes have been qualified on semiautomated hybridization machines (e.g. ThermoBrite™) Hybridization: Incubate overnight at 37 ±1 ºC in a humidified chamber. Post-Hybridization Wash: 1. Remove rubber cement seal. 2. Wash slides in Wash Buffer II (LK-103B) for 2 min at RT to slide off cover slip. 3. Wash slides in Wash Buffer I (LK-102B) for 2 min at 72 ±1 °C without agitation. Do not place more than 5 slides in the wash at one time. If more than 5 slides are to be washed, verify that the temperature on the wash solution is 72 ±1 °C 4. Note: Do not remove the coverslips from several slides before placing any of the slides in the wash
bath. Begin timing the 2 min incubation when the last slide has been added to the wash. 5. Wash slides in Wash Buffer II (LK-103B) for 1 min at room temperature without agitation. 6. Dehydrate in 70%, 85% and 100% ethanol for 1 min each. 7. Air-dry at room temperature. 8. Proceed to Counterstaining. Counterstaining: Apply 15 µl DAPI counterstain (LK-095B) and apply glass cover slip. Proceed with microscopy. Procedural recommendations: Temperature and buffer concentration (stringency) of hybridization and washing are important, as lower stringency can result in non-specific binding of the probe to other sequences and higher stringency can result in a lack of signal. Incomplete denaturation of target DNA can result in lack of signal. Material provided: LK-104B 2 x SSC solution LK-102B Wash Buffer I LK-103B Wash Buffer II LK-101B Pepsin Solution LK-095B DAPI Counterstain (0.1 µg/ml) LK-097B counterstain diluent Recommendations for Fluorescence Microscopy: For optimal visualization use a well maintained and regularly calibrated microscope equipped with a 100 W mercury lamp and a 63x or 100x fluorescent objective. Triple band-pass filters (DAPI/FITC/Texas Red or DAPI/FITC/Rhodamine) are used to view multiple colors, single band-pass filters are used for individual color visualization.
Suitable excitation and emission range for Poseidon fluorophores: PlatinumBright 415 Ex 415 ±20 nm Em 475 ±30 nm PlatinumBright 495 Ex 495 ±20 nm Em 525 ±30 nm PlatinumBright 550 Ex 546 ±12 nm Em 580 ±30 nm Material required, but not supplied: - 1% buffered formaldehyde / 1 x PBS / 20mM MgCl2 - PBS - ethanol 100%, 85% and 70% - Fixogum (LK-071A) or rubber cement - hot plate (with accurate temperature control up to 80 °C) - incubator at 37 °C - water bath with accurate temperature between 37 °C and 72 °C - variable micropipettes (1 µl - 200 µl) - fluorescence microscope equipped with suitable filters (see recommendations for Fluorescence Microscopy). Warnings and Precautions: 1. For in vitro use only. For professional use only. In case of emergencies check MSDS sheets for safety information. 2. DNA probes and hybridization buffers contain formamide which is a teratogen; do not inhale or allow skin contact. Wear gloves and a lab coat when handling DNA probes and DAPI counterstain. 3. All materials should be disposed of according to your institution’s guidelines for hospital waste disposal.
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