Post on 26-Mar-2018
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
Histology FISH Accessory Kit Step-by-Step Procedure For probes diluted in Formamide 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 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-dry2. Bake slides at 60 ˚C for 60 min to melt the paraffin3. Store at 2-8 ˚C if not used immediately
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.
Apply Probe Mix
In a fume hood apply 10 μL of Probe Mix to the center of the section. Imme-diately 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: 5 min dena- turation 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.
70% 85% 96%
3. Pepsin Digestion - RTU or Pepsin Solution
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.
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 65 °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 65 °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 for 2 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
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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
Apply 10 µL Probe
Coverslip and seal
Place on Hybridizer
Moist Humidity Strips
Start Hybridizer program 82 °C, 5 min45 °C for 14-20 h
Remove coverslip
Stringent wash, RT
Stringent wash, 65 °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 14-20 h 16 min 16 min 15 min20 min
Quick Guide