M. Fero 4/6/2006
(courtesy of Katie Rudd, Trask lab)

Materials

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
Na Azide solution (e.g. 25% stock)
Nonfat dried milk
Formamide
CHAPS or TWEEN detergent
Streptavidin-FITC (Vector)
Biotinylated goat anti-streptavidin (Vector)

Procedure

Day 1: Prepare cells and DNA

1. 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
2. Cells: Feed control cells with fresh RPMI + 15% BSA.

Day 2: Check DNA and spots cells onto slides

1. 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 H2O) 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.
2. 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:
   1. 15 μL BAC DNA (~ 1 μg)
   2. 5 μL 10x dNTP mix
   3. 5 μL enzyme
   4. 25 μL H2O
   5. Incubate at 16ºC x2 hrs, then place on ice.
   6. Run a 5 μL aliquot on a 1% agarose gel to confirm fragmentation in the 200-600 bp. (If fragments are too large the DNA then return tube to 16ºC for 1 more hr.)
   7. Stop reaction with EDTA solution. (DO THIS BEFORE ADDING CoT!)
2. Add 1 μL CoT DNA (1 μg)
3. Ethanol precipitate by adding:
   1. 200 μL TE (final vol 250 μL)
   2. 25 μL 3 M NaOAc
   3. 550 μL EtOH
   4. Incubate at -20ºC ≥ 1 hr.
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:

1. Hydrate: 2x SSC x2-5 min.
   (Optional, to reduce cytoplasm bkgd: 10 mg/mL RNAse (DNAse-free) in 2x SSC, 37ºC x30 min.)
2. Denature in 2x SSC + Formamide (50% vol), 72ºC x2-5 min.
3. 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).
4. Dry off back of slide with Kimwipe, and air dry by propping on one end.
5. Add 10 μL of probe to a region of slide with a spot of cells and cover with a 18 mm glass slip.
6. Seal edges with rubber cement using a 5 mL syringe. (Optional warm cement at 37ºC to reduce viscosity)
7. 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:
   • 2x SSC + Formamide (50% vol). 42ºC, x3.
   • 2x SSC x5 min. 42ºC, x3.
6. Blocking step:
   • Wipe off back of slide.
   • Add 100 μL block solution (4x SSC, 5% milk, 0.1% Tween-20) to the slide in 2 spots. (To store blocking reagent add 0.025% NaAzide and refrigerate at 4ºC)
   • Cover with a large glass slip for 5'.
   • Gently slide off slip.
7. Labeling step:
   • Add 100 μL of antibody (e.g. 1/400 avidin-FITC, Vector labs) diluted in blocking solution,
   • Cover with large slip. Incubate r.t. x 30 min.
8. Wash step:
   • Gently slide off cover slip.
   • Wash in 2x SSC + 0.005% CHAPS at room temp. for 5 min. (Repeat 3x)
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:
    • Biotinylated goat anti-streptavidin antibody (Vector) 0.5 mg/mL. Dilute 1/100 in
    • Streptavidin-FITC.

Tags: Fero-Histology, Fero-Lab-Protocols