Histology FISH Accessory Kit Step-by-Step Procedure For probes diluted in Ethylene Carbonate based hybridization buffer

PROCEDURE

Code K5799

Reagent Preparation Equilibration

Deparaffinization/Rehydration Prepare 2 jars with xylene or xylene substitutes RT*

Prepare 2 jars with 96 % ethanol RT

Prepare 2 jars with 70 % ethanol 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 63 ˚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 and Ethylene Carbonate hybridization buffer are not included

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-dry2. Bake slides at 60 ˚C for 60 min to melt the paraffin3. Store at 2-8 ˚C if not used immediately

Histology FISH Accessory KitVial 1: Pre-Treatment Solution (20x)Vial 2A: Pepsin, Ready-to-useVial 2B: Pepsin Diluent (10x)Vial 4: Stringent Wash Buffer (20x)Vial 5: Fluorescence Mounting MediumVial 6: Wash Buffer (20x)Item 7: Coverslip Sealant

Three Hours Thirty Minutesit’s about time

For probes in Ethylene Carobonate and FAST

hybridization

Soak slides in each of the following solutions. To finish, soak slides for 2 min in Wash Buffer.

Xylene*

5 min

Xylene*

5 min

Ethanol96%2 min

Ethanol96%2 min

Ethanol70%2 min

Ethanol70%2 min

Wash Buffer

2 min

* Or Xylene substitutes

Pre-Treatment – Microwave OvenThe microwave oven must have a boiling sensor function that can maintain temperature at 95-100 °C.

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.

Pre-Treatment – Water Bath

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.

Soak in Wash Buffer Wash the slides in Wash Buffer for 3 min at room temperature. Repeat with fresh Wash Buffer.

1. Deparaffinization & Rehydration

or

Histology FISH Accessory Kit Step-by-Step Procedure - continued

2. Heat Pre-Treatment by Microwave Oven or Water Bath

Dehydrate in Ethanol

Dehydrate tissue sections using graded ethanol series: 70%, 85%, 96% ethanol – each step for 2 min

orPepsin Digestion - Pepsin Solution

Pepsin Digestion - RTU

4. Dehydrate

Air-Dry Tissue Sections

Allow slides to air-dry completely. Take out the probe for thawing.

Apply Probe Mix

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.

Apply RTU Pepsin

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 in Pepsin Solution

Move slides to 37 °C pre-heated Pepsin Solution and incubate for 20-30 min*

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.

Incubate on Hybridizer

Incubate on Hybridizer at 37 °C for 3-5 min*

Soak in Wash Buffer

Tap off remnant Pepsin and wash in Wash Buffer for 2 x 3 min

5. Denaturation and Hybridization

Program Hybridizer

Program the Hybridizer: 10 min dena-turation at 66 °C followed by hybridiza-tion at 45 °C for 60-120 min

Moisten Strips and Place Slides

Insert moistened humidity strips in lid and place slides in the Hybridizer. Start instrument protocol.

70% 85% 96%

3. Pepsin Digestion - RTU or Pepsin Solution

Equilibrate Probe and Mix

Equilibrate Probe Mix to RT (Max. 30 min). Avoid exposure to strong light. Mix probe at 2,500 rpm on a Vortex mixer (minimum 15 s) to ensure the probe is homogenous.

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.

* Optimal incubation time depends on tissue fixation and/or thickness of specimen and should be determined by the user.

Histology FISH Accessory Kit Step-by-Step Procedure - continued

6. Stringent Wash

Apply Mounting Medium

Apply 15 μL of Fluorescence Mounting Medium containing DAPI to the target area of the slide and apply coverslips.

Storing

To minimize fading, store slides in the dark at 2-8 °C.

Reading

Slides may be read 15 min after mounting or within 7 days. Use DAPI filter and Texas Red/FITC double filter or single filters for Dako probes. Use DAPI filter and Cy3/FITC filter or single filters for SureFISH probes.

8. Mounting and Reading

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Pre-heat Stringent 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 63 °C. Measure temperature.

Remove Coverslip

Gently remove the seal and coverslip from slides, and place slides in Stringent Wash Buffer at room temperature one slide at a time.

Soak in 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.

Perfom Stringent Wash

Transfer slides to Stringent Wash Buffer jar 2 (pre-heated to 63 °C). Perform Stringent Wash for 10 min, starting count-down right after immersion of the slides into the pre-heated Stringent Wash Buffer.

Dehydrate in Ethanol 2 min x 3

Dehydrate tissue sections using graded ethanol series: 70%, 85%, 96% ethanol – each step 2 for min.

7. Dehydrate

Air-Dry Tissue Sections

Allow slides to air-dry completely.

70% 85% 96%

Histology FISH Accessory Kit Step-by-Step Procedure - continued

Notes

Three Hours Thirty Minutesit’s about time

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1. De-wax & Rehydrate

3. PepsinDigestion2. Pre-Treatment 4. Dehydrate 6. Stringent

Wash 7. Dehydrate5. Denaturation and Hybridization

Microwave oven

Pre-Treatment Solution, 95-99 °C, 10 min

Cool at RT for 15 min

Wash Buffer, 3 min X2

Or

Water Bath

Pre-Treatment Solution, 95-99 °C, 10 min

Cool at RT for 15 min

Wash Buffer, 3 min X2

Pepsin RTUTap off excess buffer

Wipe around specimen5-8 drops Pepsin RTU

Incubate, 37 °C, 3-5 min or at RT, 5-15 min

Tap off Pepsin RTU

Wash Buffer, 3 min x2

Or

Pepsin Solution

Immerse in Pepsin Solution, 37 °C, 20-30 min

Wash Buffer, 3 min x2

70% ethanol, RT, 2 min

85% ethanol, RT, 2 min

96% ethanol, RT, 2 min

Air-dry, RT, 10-20 min

Vortex RT equilibrated Probe Mix, minimum 15 s

Apply 10 µL Probe

Coverslip and seal

Place on Hybridizer

Moist Humidity Strips

Start Hybridizer program 66 °C, 10 min45 °C for 60-120 min

Remove coverslip

Stringent wash, RT

Stringent wash, 63 °C, 10 min

Wash Buffer, 3 min x2

70% ethanol, RT, 2 min

85% ethanol, RT, 2 min

96% ethanol, RT, 2 min

Air-dry, RT, 10-20 min

8. Mounting and Reading

Apply 15 µL Fluorescence Mounting Medium

Apply coverslips

Place in the dark

Read after 15 min

Xylene, 5 min x2

96% EtOH, 2 min x2

70% EtOH, 2 min x2

Wash Buffer, 2 min

31 min 9-30 min 16 min 60-120 min 16 min 16 min 15 min20 min

Quick Guide