FISH

<big>Materials</big>
20 ng BAC DNA clone grown o.n. in 5 mL LB + chloramphenicol
Autogen reagents
Slide warmer
Organics: MeOH, EtOH, formamide
Fixative (3:1 MeOH:glacial HOAc)
Biotin (or digoxigenin) nick-translation Kit (Invitrogen)
20x SSC (3 M NaCl, 0.3 M NaCitrate, pH 7.0)
TE, pH8
CoT DNA 1 µg/µL (Invitrogen)
Glass slides, 18 mm glass slips
Rubber cement
3 M NaOAc
NaAzide solution (e.g. 25% stock)
Nonfat dried milk
Formamide
CHAPS or TWEEN detergent
Streptavidin-FITC (Vector)
Biotinylated goat anti-streptavidin (Vector)

<big> <span>Procedure</span> </big>
Day 1:  Prepare cells and DNA
A. DNA:

1. Prepare BAC DNA probe on Autogen machine (see separate protocol)
2. Run 4-6 Autogen tubes per BAC. 
3. Hyb buffer:  50% formamide, 2x SSC, 10% dextran sulfate

B. Cells:   Feed control cells with fresh RPMI + 15% BSA.

Day 2 Check DNA and spots cells onto slides
A. DNA: 

1. Resuspend DNA in 50 µL/autogen tube and pool in a 1.5 mL tube. 
2. Digest with Eco RI (5 µL DNA, + 1 µL enzyme, + 1.5 µL 10x buffer, + 7.5 µL H2 O) 37ºC x 1-2 hrs.
3. Run on 0.8% agarose gel (in 0.5x TBE gel + 0.5 µg/mL ethidium Br) x 2 hrs, to check DNA quality.

B. Cells:

1. Pellet cells and wash 1x in PBS.
2. Optional:  Treat cells with hyptonic solution.
3. Pellet and resuspend in 3:1 MeOH:Glacial Acetic acid
4. Cells can be kept at -20ºC in fixative, but if so they should be washed with fresh fix on the day of use.
5. Place a wet paper towel on 37ºC hot plate.
6. Wash slides by placing in 100% MeOH in Coplin jar at room temp.
7. Wipe both sides with Kimwipe
8. Breathe on slide to moisten surface
9. Drop two spots of cells from a Pasteru pipette 6 - 12'' above slide. 
10. Place slide on moist paper towel on warmer for 1 min.
11. Move to dry part of slide warmer and leave it here o.n. to age (or place in 65ºC oven x 1hr.)

Day 3:  Label DNA and setup hybridization
1.  Setup nick-translation:
2.  Add 1 µL CoT DNA (1 µg)
3.  Ethanol precipitate by adding:
4.  Spin 5' to pellet DNA.  Wash with 70% EtOH 2x to remove salts, air dry.
5.  Resuspend DNA pellet in 55 µL of Hyb buffer. 
6.  Melt probe by incubating at 72ºC x15 min.
7.  Incubate at 37ºC x30 min. to allow CoT DNA to anneal to probe.
8.  Place probe on ice until ready to use.

Meanwhile in Coplin jars:
9.  Hydrate:  2x SSC x2-5 min.
10. (Optional, to reduce cytoplasm bkgd: 10 mg/mL RNAse (DNAse-free) in 2x SSC, 37ºC x30 min.)
11. Denature in 2x SSC + Formamide (50% vol), 72ºC x2-5 min.
12. Dehydrate:  Ice cold 70% EtOH, 85% EtOH, 95% EtOH, 100% EtOH, each 5 min.
      (Denaturing solution and EtOH washes can be reused if kept in bottles).
13. Dry off back of slide with Kimwipe, and air dry by propping on one end.
14. Add 10 µL of probe to a region of slide with a spot of cells and cover with a 18 mm glass slip.
15. Seal edges with rubber cement using a 5 mL syringe.  (Optional warm cement at 37ºC to reduce viscosity)
16. Place slides in a humid chamber o.n. at 37ºC.
 
Day 4: Washes and probe detection.
1.  Place 6 Coplin jars in 42ºC bath:  3 jars with 2x SSC + Formamide (50% vol), and 3 jars with 2x SSC only.
2.  (Reagents can be reused if kept in screw capped bottles - note order of reagents as first buffer will be the most "dirty")
3.  Warm blocking agent to room temperature (4x SSC, 5% non-fat dry milk, 0.02% Na Azide).
4.  Remove slides from humid chamber.  Remove rubber cement.  Gently slide off glass slips (dip in PBS if necessary).
5.  Wash slides: 
6.  Blocking step:
7.  Labeling step:
8.  Wash step:
9.  View on fluorescent microscope for signal.  BAC probes probably do not need further signal amplification.
10. If a second probe is being used on the same specimen then repeat the Block - Labeling - Wash steps for each (e.g. with anti-digioxigenin Texas Red). 
11. If a small probe was used or if the signal is too dim then the signal can be amplified further with additional rounds of:
    a. Biotinylated goat anti-streptavidin antibody (Vector) 0.5 mg/mL.  Dilute 1/100 in
    b. Streptavidin-FITC.