FISH ...¢ [?Δ? ]
040815 yamaichi

A. Preparation of fluorescence labeled probe

1.1 Denature the linearized template DNA (~1 μg) by boiling for 10 min.
* Template DNA to be labeled should be dissolved in D.W. (not in TE!)

1.2 Chill quickly on ice.

1.3 Add Hexanucleotide mix, fluorescence nucleotides, dNTPs mix and D.W. as follows

template DNA x μl
Hexanucleotide mix 10 μl (Roche)
fluorescence nucleotides 2.5 μl FluorX-dCTP or Cy3-dCTP (Amersham)
dNTP mix 25 μl 0.5 mM dATP, dGTP, dTTP / 0.001 mM dCTP
D.W. y μl
total 95 μl

1.4 Add 5 μl of Klenow enzyme. Mix gently by pipetting.

1.5 Incubate at 37?C for 20 hours.

2.1 Add 10 μl of 3 M NaOAC and 400 μl of 100% Ethanol to the reaction mixture.

2.2 Keep at -20?C for 30 min.

2.3 Centrifuge at 15,000 rpm for 10 min. Remove sup.

2.4 Add 700 μl of 70% Ethanol. Centrifuge for 5 min.

2.5 Carefully remove the sup by pipet.

2.6 Spin tube and carefully remove the sup by pipet.
Repeat if necessary.

2.7 Resolve in 250 μl of hybridization solution.

2.8 Sonic the probe DNA in hybridization solution for 30 sec x2.

2.9 Denature the probe DNA at 80?C for 10 min.

(optional)
2.10 To increase the specificity of the probe (especially using F probe), Add 1/10 volume of
100 μg/ml of E. coli chromosomal DNA in hybridization solution (sonicated and denatured as above).

Reagents

1. hybridization solution
50% formamide formamide 5 ml
2x SSC 20x SSC 1 ml
100 μg/ml salmon testes DNA ssDNA 1 g
D.W. 4 ml

B. Preparation of poly-L-Lysine coated slide glass

see "Preparation of poly-L-lysine coated Slides, B. All coat (for FISH) "
C. Fixation of cells [methanol : acetic acid (3:1)]

1.1 Transfer 500 μl of growing culture into 1.5-ml Eppendorf tube.
Add 500 μl of fixation solution. Keep 5 min.

1.2 Centrifuge the tube at 6,000 rpm for 5 min.
Pour off the sup and resuspend the pellet with remaining small amount (~0.1 ml) of fixation solution.

Reagents

1. fixation solution
methanol : acetic acid = 3 : 1 Methanol 75 ml
Acetic acid 25 ml

D. Denaturation and hybridization

1.3 Incubate i) a glass chamber with denaturing solution at 75?C.
ii) a glass chamber with 70% Ethanol at -20?C

1.4 Drop 10-20 μl of fixed cell suspension on a poly-L-Lysine coated glass slide.

1.5 Keep slides until cell suspension is dried (it takes few minutes).

1.5 Rinse the slide by D.W. with wash bottle. Drain water off by swinging.

1.6 Place glass slides in stainless rack.
Soak in denaturing solution at 75?C for 2 min.

1.7 Transfer to and soak in pre-chilled 70% Ethanol and keep for 5 min.

1.8 Transfer to and soak in 90% Ethanol and keep for 5 min.

1.9 Transfer to and soak in 100% Ethanol and keep for 5 min.

1.10 Air-dry with blower (by cool air).
While drying, prepare a fresh lysozyme solution.

1.11 Drop 1 ml of lysozyme solution to cover the slide. Keep 10 min.

1.12 Pour off the lysozyme solution. Drain off by swinging.

1.13 Place glass slides in stainless rack.
Soak in 1x PBS for 5 min.

1.14 Transfer to and soak in 70% Ethanol and keep for 5 min.

1.15 Transfer to and soak in 100% Ethanol and keep for 5 min.

1.16 Air-dry with blower (by cool air).

1.17 Drop 4 μl of the hybridization solution (containing fluorescence labeled probe)
onto a slide where cells are fixed.
* If you lose an area where cells were fixed on, you will breath on the slide glass and find it.

1.8 Put a cover glass and seal edges by a paper bond

1.9 Incubate at 42?C for 1-3 over-nights in moisture chamber

Reagents

1. denaturing solution
70% formamide formamide 140 ml
2x SSC 20x SSC 20 ml
D.W. 40 ml
2. lysozyme solution
2 mg/ml lysozyme in GTE buffer

*GTE; 25 mM Tris(pH8.0), 10 mM EDTA, 50 mM Glucose

E. Washing after hybridization

1.1 Incubate a glass chamber with washing solution at 37?C.

1.2 Remove sealing and a cover glass.
Place glass slides in stainless rack.
Soak in washing solution at 37?C for 15 min.

1.3 Transfer to and soak in 2x SSC and keep for 15 min.

1.4 Transfer to and soak in 1x SSC and keep for 15 min.

1.5 Transfer to and soak in 4x SSC and keep for 5 min.

1.6 Transfer to and soak in 2x SSC and keep for 5 min.

1.7 Rinse in 2x SSC with shaking up and down.

1.8 Transfer to and soak in PBS and keep for 5 min.

1.9 Transfer to and soak in D.W. and keep for 5 min.

1.10 Air-dry with blower (by cool air).
While drying, incubate the mounting solution at 37?C.

1.11 Drop 5 μl of the pre-warmed mounting solution onto a slide where cells are fixed.

1.12 Put a cover glass.

Reagents

1. washing solution
50% formamide formamide 100 ml
2x SSC 20x SSC 20 ml
D.W. 80 ml
2. mounting solution
90% Glycerol Mix 1:1
1 mg/ml p-phenylenediamine dihydrochloride 0.3 mg/ml DAPI (with DABCO) in glycerol
0.15 mg/ml DAPI 0.2% p-phenylenediamine in [2xPBS, 80% glycerol]
1x PBS

store at -20?C