Reagents Required but Not Provided
· Paraffin Pretreatment Reagent Kit (Cat No: CT-ACC112-05):
o Pretreatment Solution (50 ml): store at room temperature (RT)
o Protease Buffer (62.5 ml, pH 2.0): store at RT
o Protease (250 mg): Lyophilized, store at -20°C
· FISH Reagent Kit (Cat No: CT-ACC101-20):
o 20X Sodium Chloride-Sodium Citrate Buffer (SSC) Salt: store at RT, avoid humidity
o 4’,6-diamidino-2-phenylindole (DAPI) Counterstain: store at 4°C in the dark
o NP-40 (octylphenoxypolyethoxyethanol, or Nonidet P-40): store at RT
· Xylene: store at RT
· Ethanol (100%): store at RT
· Purified water: store at room RT
· Concentrated (12N) HCl: store at room RT
Preparation of Working Solutions
1. 20X SSC Solution (pH 7.0)
|
Reagents
|
Amount added
|
Final Concentration
|
|
SSC Salt
|
66 g
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20X
|
|
Deionized H2O (dH2O)
|
250 ml
|
|
|
TOTAL
|
250 ml
|
|
2. Protease Solution
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Reagents
|
Amount added
|
Final Concentration
|
|
Protease, lyophilized
|
250 mg
|
4 mg/ml
|
|
Protease Buffer
|
62.5 ml
|
|
|
TOTAL
|
62.5 ml
|
|
3. 90% Ethanol
|
Reagents
|
Amount added
|
Final Concentration
|
|
Ethanol (100%)
|
90 ml
|
90%
|
|
dH2O
|
10 ml
|
|
|
TOTAL
|
100 ml
|
|
4. 70% Ethanol
|
Reagents
|
Amount added
|
Final Concentration
|
|
Ethanol (100%)
|
70 ml
|
70%
|
|
dH2O
|
30 ml
|
|
|
TOTAL
|
100 ml
|
|
5. Post-hydridization Wash Solution (pH 7.0)
|
Reagents
|
Amount added
|
Final Concentration
|
|
20X SSC Solution
|
10 ml
|
2X
|
|
NP-40
|
300 µl
|
0.3%
|
|
dH2O
|
90 ml
|
|
|
TOTAL
|
100 ml
|
|
FISH Procedure for Paraffin-embedded Tissue Sections
Slide Pretreatment
1. Immerse slides in xylene at RT for 10 minutes. Repeat twice with fresh xylene each time.
2. Dehydrate slides in 100% ethanol at RT for 5 minute. Repeat once with fresh 100% ethanol.
3. Air dry slides for 2-5 minutes, if desired.
4. Immerse slides in pre-warmed Pretreatment Solution at 80°C for 10 minutes.
5. Immerse slides in purified water at RT for 3 minute.
Protease Pretreatment
1. Immerse slides in Protease Solution at 37°C for 10-60 minutes (depending on the condition of samples) and monitor the condition of cells under a light microscope.
2. Immerse slides in purified water at RT for 3 minutes.
3. Air dry slides for 2-5 minutes.
Slide Dehydration
1. Immerse slides in 70% ethanol for 3 minutes.
2. Immerse slides in 90% ethanol for 3 minutes.
3. Immerse slides in 100% ethanol for 3 minutes.
4. Air dry slides.
Probe Preparation
1. Pre-warm the probe at RT for 20-30 minutes.
2. Briefly vortex and spin down the probe.
Co-denaturation & Hybridization
1. Apply 10 μl of the probe on each hybridization area and cover with a 22 mm x 22 mm coverslip. Seal coverslip(s) with rubber cement.
2. Co-denature slides with probe at 72°C for 5 minutes.
3. Place slides in a pre-warmed humidified hybridization chamber and incubate slides at 37°C overnight (16 hours).
Post-hybridization Wash
1. Mark each hybridization area on the back of the slides with a diamond-tip pen.
2. Carefully remove rubber cement.
3. Immerse slides in Post-hybridization Wash Solution at RT to loosen the coverslips. Shake gently to allow the coverslips to detach unaided; do not pull the coverslips off.
4. Immerse slides in pre-warmed Post-hybridization Wash Solution at 72°C for 2 minutes.
Slide Dehydration
1. Immerse slides in 70% ethanol for 2 minutes.
2. Immerse slides in 90% ethanol for 2 minutes.
3. Immerse slides in 100% ethanol for 2 minutes.
4. Air dry slides in the dark.
Visualization
1. Apply DAPI counterstain and cover slides with coverslips.
Examine slides under a fluorescence microscope with proper filter sets. |
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