PROCEDURE Histology FISH Accessory Kit Step-by-Step Procedure Code K5799 For probes diluted in Formamide based hybridization buffer Reagent Preparation Equilibration Deparaffinization/Rehydration Prepare 2 jars with xylene or xylene substitutes RT* Prepare 2 jars with 96 % ethanol Prepare 2 jars with 70 % ethanol RT RT Wash Buffer (Vial 6) Dilute Vial 6 1:20 using distilled or deionized water (RT) RT Pre-Treatment Solution (Vial 1) Dilute Vial 1 1:20 using distilled or deionized water (RT) If microwave oven: RT, if water bath: 95-99 C Pepsin RTU (Vial 2A) - 2-8 C Pepsin Solution (Vial 2A+2B) Dilute Vial 2B using distilled or deionized water then add Vial 2A, dilute 1+8+1 37 C Dehydration Prepare 3 jars with 70%, 85% and 96% ethanol RT Stringent Wash Buffer (Vial 4) Dilute Vial 4 1:20 using distilled or deionized water (RT) RT and 65 C ± 2 C Fluorescence Mounting Medium (Vial 5) - 2-25 C Coverslip Sealant - 2-25 C *RT = Room Temperature (20-25 C) NOTE: Probe Mix is not included For probes in formamide and Overnight hybridization Slide Preparation Only tissue preserved in neutral buffered formalin (fixation time 18-24 hours) and paraffin-embedded is suitable for use. The optimal thickness of tissue sections varies with tissue and probe type. In general the optimal tissue section thickness is 2-5 μm for Translocation Probe applications and 3-6 μm for Gene Copy Number Probe applications. 1. Cut sections on water bath, collect on slides and air-dry 2. Bake slides at 60 C for 60 min to melt the paraffin 3. Store at 2-8 C if not used immediately Histology FISH Accessory Kit Vial 1: Pre-Treatment Solution (20x) Vial 2A: Pepsin, Ready-to-use Vial 2B: Pepsin Diluent (10x) Vial 4: Stringent Wash Buffer (20x) Vial 5: Fluorescence Mounting Medium Vial 6: Wash Buffer (20x) Item 7: Coverslip Sealant
1. Deparaffinization & Rehydration Soak slides in each of the following solutions. To finish, soak slides for in Wash Buffer. 2. Heat Pre-Treatment by Microwave Oven or Water Bath Xylene* 5 min Xylene* 5 min * Or Xylene substitutes 96% 96% 70% 70% Wash Buffer Pre-Treatment Microwave Oven The microwave oven must have a boiling sensor function that can maintain temperature at 95-100 C. or Pre-Treatment Water Bath Pre-Treatment in Microwave Oven Incubate for 10 min with the boiling sensor function Cool for 15 Minutes Remove the jar from the microwave oven. Take off the lid and let the slides cool in the solution for 15 min at room temperature. Preheat Pre-Treatment Solution in Water Bath Place a jar filled with Pre-Treatment Solution in a water bath and heat to 95-99 C. Measure temperature. Incubate for 10 Minutes at 95-99 C Do not start count-down before Pre- Treatment Solution including slides is minimum 95 C. Cool for 15 Minutes Remove the jar from the water bath. Take off the lid and let the slides cool in the solution for 15 min at room temperature. Histology FISH Accessory Kit Step-by-Step Procedure - continued Soak in Wash Buffer Wash the slides in Wash Buffer for 3 min at room temperature. Repeat with fresh Wash Buffer.
3. Pepsin Digestion - RTU or Pepsin Solution Pepsin Digestion - RTU * Optimal incubation time depends on tissue fixation and/or thickness of specimen and should be determined by the user. Pepsin Ready-to-Use Tap off excess buffer and wipe around the specimen using a lint free paper towel. Tissue sections must not dry out. Apply RTU Pepsin or Pepsin Digestion - Pepsin Solution Apply 5-8 drops of cold RTU Pepsin. Make sure the tissue is covered. Incubate at RT for 5-15 min* or move on to next step on Hybridizer. Incubate on Hybridizer Incubate on Hybridizer at 37 C for 3-5 min* 4. Dehydrate 70% 85% 96% Incubate in Pepsin Solution Move slides to 37 C pre-heated Pepsin Solution and incubate for 20-30 min* 5. Denaturation and Hybridization Soak in Wash Buffer Tap off remnant Pepsin and wash in Wash Buffer for 2 x 3 min Dehydrate in Dehydrate tissue sections using graded ethanol series: 70%, 85%, 96% ethanol each step for Air-Dry Tissue Sections Allow slides to air-dry completely. Apply Probe Mix In a fume hood apply 10 μl of Probe Mix to the center of the section. Immediately place the coverslip allow it to spread evenly and avoid air bubbles. Seal Coverslip Seal the coverslip with Coverslip Sealant all around the periphery. Coverslip Sealant should overlap the coverslip and the slide and cover the entire edge. Program Hybridizer Program the Hybridizer: 5 min denaturation at 82 C and overnight (14-20 h) hybridization at 45 C (pre-set protocol). Moisten Strips and Place Slides Insert moistened humidity strips in lid and place slides in the Hybridizer. Start instrument protocol. Histology FISH Accessory Kit Step-by-Step Procedure - continued
6. Stringent Wash Pre-heat Stringent Wash Buffer Remove Coverslip Perfom Stringent Wash Soak in Wash Buffer Fill two jars with Stringent Wash Buffer. Place jar 1 with Stringent Wash Buffer at room temperature. Place jar 2 with a lid in a water bath and pre-heat to 65 C. Measure temperature. Gently remove the seal and coverslip from slides, and place slides in Stringent Wash Buffer at room temperature one slide at a time. Transfer slides to Stringent Wash Buffer jar 2 (pre-heated to 65 C). Perform Stringent Wash for 10 min, starting count-down right after immersion of the slides into the pre-heated Stringent Wash Buffer. Remove slides from Stringent Wash Buffer and place them in Wash Buffer for 3 min at room temperature. Repeat with fresh Wash Buffer. 7. Dehydrate 8. Mounting and Reading 70% 85% 96% Dehydrate in x 3 Air-Dry Tissue Sections Apply Mounting Medium Storing Reading Dehydrate tissue sections using graded ethanol series: 70%, 85%, 96% ethanol each step for. Allow slides to air-dry completely. Apply 15 μl of Fluorescence Mounting Medium containing DAPI to the target area of the slide and apply coverslips. To minimize fading, store slides in the dark at 2-8 C. Slides may be read 15 min after mounting or within 7 days. Use DAPI filter and Texas Red/FITC double filter or single filters. Service, Support and Training Dako is committed to ensuring that our customers feel confident using our products through training, demonstrations, service and support. With an extensive geographic presence, our local staff is always within easy reach. No matter where you are in the world, our Customer Care team of trainers, instrument and technical support experts, applications specialists and customer service representatives are just a visit, phone call, or e-mail away. Histology FISH Accessory Kit Step-by-Step Procedure - continued
Notes
Quick Guide 1. De-wax & Rehydrate 2. Pre-Treatment 3. Pepsin 4. Dehydrate Digestion 5. Denaturation and Hybridization 6. Stringent Wash 7. Dehydrate 8. Mounting and Reading Xylene, 5 min x2 96% EtOH, x2 70% EtOH, x2 Wash Buffer, Microwave oven Pre-Treatment Solution, 95-99 C, 10 min Cool at RT for 15 min X2 Pepsin RTU Tap off excess buffer Wipe around specimen 5-8 drops Pepsin RTU Incubate, 37 C, 3-5 min or at RT, 5-15 min 70% ethanol, RT, 85% ethanol, RT, 96% ethanol, RT, Air-dry, RT, 10-20 min Apply 10 µl Probe Coverslip and seal Place on Hybridizer Moist Humidity Strips Remove coverslip Stringent wash, RT Stringent wash, 65 C, 10 min x2 70% ethanol, RT, 85% ethanol, RT, 96% ethanol, RT, Air-dry, RT, 10-20 min Apply 15 µl Fluorescence Mounting Medium Apply coverslips Place in the dark Read after 15 min Tap off Pepsin RTU x2 Start Hybridizer program 82 C, 5 min 45 C for 14-20 h Or Or Water Bath Pre-Treatment Solution, 95-99 C, 10 min Pepsin Solution Immerse in Pepsin Solution, 37 C, 20-30 min Cool at RT for 15 min x2 X2 20 min 31 min 9-30 min 16 min 14-20 h 16 min 16 min 15 min Corporate Headquarters Denmark +45 44 85 95 00 www.dako.com Represented in more than 100 countries Australia +61 3 9357 0892 Austria +43 1 408 43 34 0 Belgium +32 (0) 16 38 72 20 Brazil +55 11 50708300 Canada +1 905 335 3256 China +86 21 3612 7091 Denmark +45 44 85 97 56 Finland +358 9 348 73 950 France +33 1 64 53 61 44 Germany +49 40 69 69 470 Ireland +353 1 479 0568 Italy +39 02 58 078 1 Japan +81 3 5802 7211 Korea +82 2 402 6775 The Netherlands +31 20 42 11 100 Norway +47 23 14 05 40 Poland +48 58 661 1879 Spain +34 93 499 05 06 Sweden +46 8 556 20 600 Switzerland +41 41 760 11 66 United Kingdom +44 (0)1 353 66 99 11 United States of America +1 805 566 6655 29072 31OCT13