Cytology FISH Accessory Kit Code/ Code/ Code-Nr. K5499

2nd edition/ 2ème édition/ 2. Ausgabe

For fluorescence in situ hybridization (FISH) on cytology specimens. The kit contains reagents sufficient for 20 tests.

Pour l'hybridation in situ fluorescente (FISH) sur des échantillons cytologiques. La trousse contient assez de réactifs pour 20 tests.

Für an Zytologieproben durchzuführende In-situ-Hybridisierung unter direkter Fluoreszenz (FISH). Der Kitinhalt ist für 20 Tests ausreichend.

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Contents/ Sommaire/ Inhalt Page/ Page/ Seite ENGLISH Intended Use ................................................................................................................................ 5 Introduction................................................................................................................................... 5 Reagents ...................................................................................................................................... 5 Materials provided.................................................................................................................... 5 Materials required but not provided .......................................................................................... 5 Precautions .................................................................................................................................. 6 Storage......................................................................................................................................... 7 INSTRUCTIONS FOR USE A. Reagent Preparation ................................................................................................................ 8 A.1 Wash Buffer ..................................................................................................................... 8 A.2 3.7% formaldehyde........................................................................................................... 8 A.3 Ethanol series................................................................................................................... 8 A.4 Stringency Buffer .............................................................................................................. 8 B. Staining Procedure................................................................................................................... 8 B.1 Procedural notes .............................................................................................................. 8 B.2 Staining protocol............................................................................................................... 8 Quality Control............................................................................................................................ 10 Interpretation of Results ............................................................................................................. 10 References ................................................................................................................................. 10 Troubleshooting.......................................................................................................................... 11 Appendix 1 ................................................................................................................................. 12 Explanation of symbols............................................................................................................... 34

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FRANÇAIS Intérêt ......................................................................................................................................... 13 Introduction................................................................................................................................. 13 Réactifs ...................................................................................................................................... 13 Matériaux fournis ................................................................................................................... 13 Matériaux requis mais non fournis ......................................................................................... 14 Précautions d'emploi .................................................................................................................. 14 Stockage .................................................................................................................................... 16 INSTRUCTIONS D'UTILISATION A. Préparation des réactifs ......................................................................................................... 16 A.1 Tampon de lavage .......................................................................................................... 16 A.2 Formaldéhyde à 3,7 %.................................................................................................... 16 A.3 Série de bains d'éthanol ................................................................................................. 16 A.4 Tampon de lavage stringent ........................................................................................... 16 B. Procédure d'immunomarquage .............................................................................................. 17 B.1 Notes procédurales......................................................................................................... 17 B.2 Protocole de marquage .................................................................................................. 17 Contrôle de qualité ..................................................................................................................... 18 Interprétation des résultats ......................................................................................................... 19 Références ................................................................................................................................. 19 Dépannage................................................................................................................................. 20 Annexe 1 .................................................................................................................................... 21 Légende des symboles............................................................................................................... 34

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DEUTSCH Zweckbestimmung...................................................................................................................... 23 Einleitung.................................................................................................................................... 23 Reagenzien ................................................................................................................................ 23 Packungsinhalt....................................................................................................................... 23 Zusätzlich benötigte Reagenzien und Zubehör (außerhalb des Lieferumfangs)..................... 24 Hinweise und Vorsichtsmaßnahmen........................................................................................... 24 Lagerung .................................................................................................................................... 27 GEBRAUCHSANLEITUNG A. Reagenzienvorbereitung ........................................................................................................ 27 A.1 Waschpuffer (Wash Buffer) ............................................................................................ 27 A.2 3,7%iges Formaldehyd ................................................................................................... 27 A.3 Ethanol-Reihe................................................................................................................. 27 A.4 Stringency Buffer ............................................................................................................ 27 B. Färbeprozedur........................................................................................................................ 28 B.1 Verfahrenshinweise ........................................................................................................ 28 B.2 Färbungsprotokoll........................................................................................................... 28 Qualitätskontrolle........................................................................................................................ 29 Interpretation der Ergebnisse ..................................................................................................... 30 Literatur ...................................................................................................................................... 30 Fehlerbehebung ......................................................................................................................... 31 Anhang 1 .................................................................................................................................... 32 Erläuterung der Symbole ............................................................................................................ 34

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ENGLISH Intended Use For in vitro diagnostic use. Cytology FISH Accessory Kit is intended for use in the fluorescence in situ hybridization (FISH) technique on cytology specimens.

Introduction The kit is employed in a two-day procedure. The first day, a pre-treatment of the cytology specimens (provided by the user) is performed using 3.7% formaldehyde for 2 minutes, before dehydration using ethanol. Following the pre-treatment, user-provided FISH probes are applied and specimens are sealed with Coverslip Sealent, before denaturation and hybridization overnight. The second day, a Stringency Wash is performed, which ensure removal of unbound and unspecific bound FISH probe, before dehydration with ethanol. Finally, the specimens are mounted with Fluorescence Mounting Medium containing a blue fluorescence counterstain and coverslipped. Results are interpreted using a fluorescence microscope equipped with appropriate filter (see Appendix 1).

Reagents Materials provided The materials listed below are sufficient for 20 tests (a test is defined as one 18 mm x 18 mm target area). Vial 1 Wash Buffer (x 20) 500 mL, 20 x concentrated Tris/HCl buffer. Vial 2 Stringency Buffer (x 20) 150 mL, 20 x concentrated SSC (saline-sodium citrate) buffer with a detergent. Vial 3 Fluorescence Mounting Medium 300 µL Ready-to-use fluorescence mounting medium with a blue counter stain. Item 4 Coverslip Sealant 1 tube Ready-to-use solution for removable sealing of coverslip. NOTE: All reagents are formulated specifically for use with this kit.

Materials required but not provided Laboratory reagents Distilled or deionized water Ethanol, 96% Formaldehyde, 37% (128170-001)

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Laboratory equipment Adjustable pipettes Calibrated partial immersion thermometer (range 37-100 °C) Calibrated surface thermometer (range 37-100 °C) Coverslips (18 mm x 18 mm) Forceps Fume hood Heating block or hybridization oven for denaturation (82 (±2) °C) Humid hybridization chamber Hybridization oven or incubator for overnight hybridization (45 (±2) °C) Slides, Dako Silanized Slides, code No. S 3003, poly-L-lysine-coated slides, or superfrost slides Staining jars or baths Timer (capable of 2-15 minute intervals) Water bath with lid (capable of maintaining 65 (±2) °C to 99 °C) Microscope equipment and accessories Filters for fluorescence microscope: DAPI filter and suitable filters for the used fluorochrome, e.g. FITC/Texas Red double filter, or FITC and Texas Red mono filters for Dako FISH Probes - see Appendix 1 for details Fluorescence microscope with a 100 watt mercury lamp is recommended for Dako FISH Probes Microscope slide folder (cardboard tray for 20 slides with hinged cover or similar)

Precautions 1. 2.

For professional users. Vial 1, Wash Buffer (20x), contains ≥10-