12. 2% agarose gel: in a beaker, weigh 2 g of agarose, add 100 mL
1Â TAE. Dissolve in microwave and pour in casting system.
13. 3 M sodium acetate pH 5.2. For 100 mL, in a beaker, dissolve
24.6 g NaAc anhydrous in H 2 O and adjust pH at 5.2 with
glacial acetic acid and complete up to 100 mL with H 2 O in a
graduated cylinder.
14. 75% ethanol.
15. Cold 70% and 100% ethanol stored at À20
C.
16. 0.2 M sodium bicarbonate (NaHCO 3 ). For 100 mL: in a
beaker, dissolve 1.68 g NaHCO 3 in H 2 O and complete with
H 2 O up to 100 mL with a graduated cylinder.
17. 0.5 M Tris–HCl pH 8.5. For 100 mL, in a beaker, dissolve
6.05 g Tris base in approximately 90 mL H 2 O. Add 37% HCl
very slowly up to pH ¼ 8.5 (see Note 4). Complete with H 2 O
up to 100 mL in a graduated cylinder.
18. 10 mM Tris–HCl pH 8.5. For 100 mL, combine 1 mL of
0.5 M Tris–HCl pH 8.5 with 99 mL H 2 O.
19. Amine reactive dyes: we used Alexa Fluor Succinimidyl dyes,
488, 555 and 647 from Invitrogen Molecular Probes.
20. Anhydrous DMSO.
2.4.2 DNA-FISH Labeling
1. 4% paraformaldehyde (PFA) in 1Â PBS. Always prepare fresh.
For 10 mL of fixation solution: combine 2.5 mL 16% paraformaldehyde and 7.5 mL 1Â PBS and mix vigorously (see
Note 16).
2. RNase A stock solution at 2 mg/mL in H 2 O: for 50 mL,
dissolve 100 mg RNase A in 50 mL H 2 O. Filter sterilize
using a 0.2 μm filter. Store aliquots as 10 mL aliquots at
À20
C.
3. RNase A at 0.2 mg/mL in 2Â SSC. Dilute tenfold RNase A
stock solution in 2Â SSC and store 10 mL aliquots at À20
C.
4. 20Â SSC.
5. 4Â SSC in H 2 O. For 50 mL: combine 10 mL of 20Â SSC stock
solution with 40 mL H 2 O.
6. 2Â SSC in H 2 O. Dilute 20Â SSC tenfold in H 2 O.
7. 0.1Â SSC in H 2 O. For 50 mL: combine 2.5 mL of 2Â SSC
with 37.5 mL H 2 O.
8. 4Â SSC/0.2% Tween20. For 50 mL: dilute 100 μL of
Tween20 in 50 mL of 4Â SSC (see Note 12).
9. 0.5% (v/v) Triton X-100 in 1Â PBS: for 100 mL, dilute 500 μL
Triton X-100 in 100 mL 1Â PBS (see Note 17).
10. 0.01% (v/v) Triton X-100 in 1Â PBS: for 100 mL, dilute 2 mL
of 0.5% (v/v) Triton X-100 in 198 mL 1Â PBS.
EMT Studied by Molecular Biology and DNA-FISH in Human Cells
359
1Â TAE. Dissolve in microwave and pour in casting system.
13. 3 M sodium acetate pH 5.2. For 100 mL, in a beaker, dissolve
24.6 g NaAc anhydrous in H 2 O and adjust pH at 5.2 with
glacial acetic acid and complete up to 100 mL with H 2 O in a
graduated cylinder.
14. 75% ethanol.
15. Cold 70% and 100% ethanol stored at À20
C.
16. 0.2 M sodium bicarbonate (NaHCO 3 ). For 100 mL: in a
beaker, dissolve 1.68 g NaHCO 3 in H 2 O and complete with
H 2 O up to 100 mL with a graduated cylinder.
17. 0.5 M Tris–HCl pH 8.5. For 100 mL, in a beaker, dissolve
6.05 g Tris base in approximately 90 mL H 2 O. Add 37% HCl
very slowly up to pH ¼ 8.5 (see Note 4). Complete with H 2 O
up to 100 mL in a graduated cylinder.
18. 10 mM Tris–HCl pH 8.5. For 100 mL, combine 1 mL of
0.5 M Tris–HCl pH 8.5 with 99 mL H 2 O.
19. Amine reactive dyes: we used Alexa Fluor Succinimidyl dyes,
488, 555 and 647 from Invitrogen Molecular Probes.
20. Anhydrous DMSO.
2.4.2 DNA-FISH Labeling
1. 4% paraformaldehyde (PFA) in 1Â PBS. Always prepare fresh.
For 10 mL of fixation solution: combine 2.5 mL 16% paraformaldehyde and 7.5 mL 1Â PBS and mix vigorously (see
Note 16).
2. RNase A stock solution at 2 mg/mL in H 2 O: for 50 mL,
dissolve 100 mg RNase A in 50 mL H 2 O. Filter sterilize
using a 0.2 μm filter. Store aliquots as 10 mL aliquots at
À20
C.
3. RNase A at 0.2 mg/mL in 2Â SSC. Dilute tenfold RNase A
stock solution in 2Â SSC and store 10 mL aliquots at À20
C.
4. 20Â SSC.
5. 4Â SSC in H 2 O. For 50 mL: combine 10 mL of 20Â SSC stock
solution with 40 mL H 2 O.
6. 2Â SSC in H 2 O. Dilute 20Â SSC tenfold in H 2 O.
7. 0.1Â SSC in H 2 O. For 50 mL: combine 2.5 mL of 2Â SSC
with 37.5 mL H 2 O.
8. 4Â SSC/0.2% Tween20. For 50 mL: dilute 100 μL of
Tween20 in 50 mL of 4Â SSC (see Note 12).
9. 0.5% (v/v) Triton X-100 in 1Â PBS: for 100 mL, dilute 500 μL
Triton X-100 in 100 mL 1Â PBS (see Note 17).
10. 0.01% (v/v) Triton X-100 in 1Â PBS: for 100 mL, dilute 2 mL
of 0.5% (v/v) Triton X-100 in 198 mL 1Â PBS.
EMT Studied by Molecular Biology and DNA-FISH in Human Cells
359