Reagents Required but Not Provided
· FISH Reagent Kit (Cat No: CT-ACC101-20):
o 20X SSC Salt: store at RT, avoid humidity
o DAPI Counterstain: store at 4°C in the dark
o NP-40: store at RT
· Pepsin (Lyophilized): store at -20°C or below
· Hydrochloric acid (1N): store at RT
· Formaldehyde (37%): store at RT
· 10X Phosphate-buffered Saline (PBS) Solution: store at RT
· Ethanol (100%): store at RT
Preparation of Working Solutions
1. 20X SSC Solution (pH 7.0)
|
Reagents
|
Amount added
|
Final Concentration
|
|
SSC Salt
|
66 g
|
20X
|
|
dH2O
|
250 ml
|
|
|
TOTAL
|
250 ml
|
|
2. Pepsin Stock Solution
|
Reagents
|
Amount added
|
Final Concentration
|
|
Pepsin, lyophilized
|
100 mg
|
100 mg/ml
|
|
dH2O
|
1 ml
|
|
|
TOTAL
|
1 ml
|
|
Note: Keep on ice. Make 20 µl aliquots, store at -20°C.
3. Pepsin Working Solution
|
Reagents
|
Amount added
|
Final Concentration
|
|
HCl (1N)
|
1 ml
|
0.01N
|
|
Pepsin Stock Solution
|
20 µl
|
0.02 µg/µl
|
|
dH2O
|
99 ml
|
|
|
TOTAL
|
100 ml
|
|
4. 2X SSC Solution
|
Reagents
|
Amount added
|
Final Concentration
|
|
20X SSC Solution
|
10 ml
|
2X
|
|
dH2O
|
90 ml
|
|
|
TOTAL
|
100 ml
|
|
5. 1X PBS Solution
|
Reagents
|
Amount added
|
Final Concentration
|
|
10X PBS Solution
|
10 ml
|
1X
|
|
dH2O
|
90 ml
|
|
|
TOTAL
|
100 ml
|
|
6. Formaldehyde Solution
|
Reagents
|
Amount added
|
Final Concentration
|
|
Formaldehyde (37%)
|
2.7 ml
|
1%
|
|
10X PBS Solution
|
10 ml
|
1X
|
|
dH2O
|
89 ml
|
|
|
TOTAL
|
100 ml
|
|
7. 90% Ethanol
|
Reagents
|
Amount added
|
Final Concentration
|
|
Ethanol (100%)
|
90 ml
|
90%
|
|
dH2O
|
10 ml
|
|
|
TOTAL
|
100 ml
|
|
8. 70% Ethanol
|
Reagents
|
Amount added
|
Final Concentration
|
|
Ethanol (100%)
|
70 ml
|
70%
|
|
dH2O
|
30 ml
|
|
|
TOTAL
|
100 ml
|
|
9. Post-hybridization Wash Solution 1
|
Reagents
|
Amount added
|
Final Concentration
|
|
20X SSC Solution
|
2 ml
|
0.4X
|
|
NP-40
|
300 µl
|
0.3%
|
|
dH2O
|
98 ml
|
|
|
TOTAL
|
100 ml
|
|
10. Post-hybridization Wash Solution 2
|
Reagents
|
Amount added
|
Final Concentration
|
|
20X SSC Solution
|
10 ml
|
2X
|
|
NP-40
|
100 µl
|
0.1%
|
|
dH2O
|
90 ml
|
|
|
TOTAL
|
100 ml
|
|
FISH Procedure for Cytology
Slide Preparation
1. Equilibrate slides in 2X SSC Solution at RT for 2 minutes.
2. Immerse slides in pre-warmed Pepsin Working Solution at 37°C for 1-10 minutes (depending on the condition of samples) and monitor the condition of cells under a light microscope.
3. Wash slides in 1X PBS Solution at RT for 5 minutes.
4. Post-fix slides in Formaldehyde Solution at RT for 5 minutes.
5. Wash slides in 1X PBS Solution at RT for 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 probes at RT for 20~30 minutes.
2. Briefly vortex and spin down probes.
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 2 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 hybridization area on the back of the slides with a diamond-tip pen.
2. Carefully remove the rubber cement.
3. Soak slide in 2X SSC Solution at RT to loosen coverslip(s). Do not pull off coverslip(s).
4. Immerse slides in pre-warmed Post-hybridization Wash Solution 1 at 72°C for 1 minute.
5. Immerse slides in Post-hybridization Wash Solution 2 at RT for 2 minutes.
6. Air dry slides.
Visualization
1. Apply DAPI counterstain, cover with coverslip(s).
Examine slides under a fluorescence microscope with proper filter sets. |
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