7. Add 5 μL of dextran sulfate mix and shake on a thermomixer at
1000 rpm for further 10 min under an aluminum foil (see Note
64).
3.3.4 Coverslip Assembly
for Hybridization
1. Drop up to 30 μL of the DNA-FISH probe mixture (for
localization of up to three different loci) on a glass slide (see
Note 65).
2. Take the glass coverslip with the fixed cells (laying in the 50%
formamide solution) using tweezers, quickly but gently wipe
the lower side and drain the excess of fluid from the upper side
with a tissue (see Note 66).
3. Return the coverslip on the glass slide to lay the upper side of
the coverslip with the DNA-FISH probe mixture droplet and
gently dry the excess of fluid around the coverslip.
4. Seal the coverslip with rubber cement (see Note 67) and let the
slide/coverslip set-up in the dark.
3.3.5 Denaturation
and Hybridization
1. Humidify the hybridization chamber and place the glass slide/
coverslip set-up on the heating block (the coverslip facing up
onto the heating block).
2. For MCF10A cells, denaturation conditions are 75
C for
4 min and hybridization occurs at 37
C overnight.
3.3.6 Washing FISH
Samples
1. Heat up 2Â SSC to 45
C and 0.1Â SCC up to 60
C in water
baths.
2. Fill a 6-well plate (or a 35 mm petri dish depending on the
number of coverslips to wash), with 2 mL 2Â SSC heaten at
45
C.
3. Gently remove the rubber cement with a tweezer and transfer
the coverslip in the well, cells face up (see Note 68).
4. Remove the supernatant and wash with 2 mL of 45
C 2Â SSC
for 3 min. Repeat three times.
5. Wash with 2 mL of 60
C 0.1Â SSC for 3 min. Repeat three
times.
3.3.7 Hoechst Staining
1. Add 2 mL of Hoechst 1/5000 dilution in 1Â PBS and leave at
room temperature and protected from light for 10 min.
2. Wash with 1Â PBS for 10 min. Repeat twice.
3. Optional: Sample can be stored in 1Â PBS at 4
C for up to
2 weeks.
3.3.8 Sample Mounting
for Microscopy Acquisition
1. Fill the cavity of the concave slide with 80 μL of 1Â PBS.
2. Place the coverslip on the cavity.
374
Noe ´ mie Kempf et al.
1000 rpm for further 10 min under an aluminum foil (see Note
64).
3.3.4 Coverslip Assembly
for Hybridization
1. Drop up to 30 μL of the DNA-FISH probe mixture (for
localization of up to three different loci) on a glass slide (see
Note 65).
2. Take the glass coverslip with the fixed cells (laying in the 50%
formamide solution) using tweezers, quickly but gently wipe
the lower side and drain the excess of fluid from the upper side
with a tissue (see Note 66).
3. Return the coverslip on the glass slide to lay the upper side of
the coverslip with the DNA-FISH probe mixture droplet and
gently dry the excess of fluid around the coverslip.
4. Seal the coverslip with rubber cement (see Note 67) and let the
slide/coverslip set-up in the dark.
3.3.5 Denaturation
and Hybridization
1. Humidify the hybridization chamber and place the glass slide/
coverslip set-up on the heating block (the coverslip facing up
onto the heating block).
2. For MCF10A cells, denaturation conditions are 75
C for
4 min and hybridization occurs at 37
C overnight.
3.3.6 Washing FISH
Samples
1. Heat up 2Â SSC to 45
C and 0.1Â SCC up to 60
C in water
baths.
2. Fill a 6-well plate (or a 35 mm petri dish depending on the
number of coverslips to wash), with 2 mL 2Â SSC heaten at
45
C.
3. Gently remove the rubber cement with a tweezer and transfer
the coverslip in the well, cells face up (see Note 68).
4. Remove the supernatant and wash with 2 mL of 45
C 2Â SSC
for 3 min. Repeat three times.
5. Wash with 2 mL of 60
C 0.1Â SSC for 3 min. Repeat three
times.
3.3.7 Hoechst Staining
1. Add 2 mL of Hoechst 1/5000 dilution in 1Â PBS and leave at
room temperature and protected from light for 10 min.
2. Wash with 1Â PBS for 10 min. Repeat twice.
3. Optional: Sample can be stored in 1Â PBS at 4
C for up to
2 weeks.
3.3.8 Sample Mounting
for Microscopy Acquisition
1. Fill the cavity of the concave slide with 80 μL of 1Â PBS.
2. Place the coverslip on the cavity.
374
Noe ´ mie Kempf et al.