9. Centrifuge the cells for 4 min at 1300 Â g at 4
C and wash
them once with 500 μL of ice-cold buffer B. Do not vortex but
resuspend the cells with the pipet. Cells are fragile after the
Lyticase treatment.
10. Resuspend the cells in 900 μL of ice-cold buffer B. Drop
200 μL on each poly-L-lysine-treated coverslip. With this
amount of cultures 5–6 coverslips can be prepared for each
sample that can be then hybridized with different probes or
antibodies.
11. Incubate for 30–90 min at 4
C to allow the cells to adhere to
the coverslips.
12. Wash once each well with 2 mL ice-cold buffer B. Gently
aspirate the buffer and add 2 mL of cold 70% ethanol. Seal
the plate with parafilm and store at À20
C (see Note 8).
3.3 Hybridization
1. Move the coverslips for hybridization into a new 12-well plate.
Rehydrate the cells by two washes with 2 mL of 2Â SSC, each
for 5 min at RT (see Note 9).
2. Incubate the coverslips in 2 mL of pre-hybridization solution
for 30 min at RT (see Note 10).
3. For each coverslip, combine 0.125 μL of the original stock of
probes for the mRNA of interest (stock concentration ¼ 25 μM)
with 5 μL of the DNA/RNA competitor. Lyophilize in a
Vacufuge at 45
C (see Note 11).
4. 12.5 μL of solution F coverslips: Heat at 95
C for 2 min. Let
the solution cool at RT for about 5 min. Keep probes in the
dark to prevent photobleaching.
5. Add 12.5 μL of solution H per each coverslip. The resulting
hybridization solution in the well (25 μL) now contains
125 nM probe mixture and 10% formamide.
6. Cover the bottom of a 150 mm petri dish with parafilm and
tape it to the bottom to keep the surface flat (Fig. 2a).
7. Transfer 23 μL of the hybridization solution from step 5 onto
the parafilm, one drop for each coverslip. By using the forceps,
take the coverslip from each well, remove from each coverslip
the leftover pre-hybridization solution using a Kimtech tissue,
and place each coverslip face down onto the prepared hybridization drop. Place a small container (e.g., the cap of a 15 mL
plastic tube) toward the edge of the Petri dish and fill it with
DDW. Cover and seal the petri dish with parafilm to create a
hybridization chamber. Incubate in the dark at 37
C for 3 h.
Incubate at 37
C the pre-hybridization solution that you will
use for the following washes (step 8).
58
Evelina Tutucci and Robert H. Singer
C and wash
them once with 500 μL of ice-cold buffer B. Do not vortex but
resuspend the cells with the pipet. Cells are fragile after the
Lyticase treatment.
10. Resuspend the cells in 900 μL of ice-cold buffer B. Drop
200 μL on each poly-L-lysine-treated coverslip. With this
amount of cultures 5–6 coverslips can be prepared for each
sample that can be then hybridized with different probes or
antibodies.
11. Incubate for 30–90 min at 4
C to allow the cells to adhere to
the coverslips.
12. Wash once each well with 2 mL ice-cold buffer B. Gently
aspirate the buffer and add 2 mL of cold 70% ethanol. Seal
the plate with parafilm and store at À20
C (see Note 8).
3.3 Hybridization
1. Move the coverslips for hybridization into a new 12-well plate.
Rehydrate the cells by two washes with 2 mL of 2Â SSC, each
for 5 min at RT (see Note 9).
2. Incubate the coverslips in 2 mL of pre-hybridization solution
for 30 min at RT (see Note 10).
3. For each coverslip, combine 0.125 μL of the original stock of
probes for the mRNA of interest (stock concentration ¼ 25 μM)
with 5 μL of the DNA/RNA competitor. Lyophilize in a
Vacufuge at 45
C (see Note 11).
4. 12.5 μL of solution F coverslips: Heat at 95
C for 2 min. Let
the solution cool at RT for about 5 min. Keep probes in the
dark to prevent photobleaching.
5. Add 12.5 μL of solution H per each coverslip. The resulting
hybridization solution in the well (25 μL) now contains
125 nM probe mixture and 10% formamide.
6. Cover the bottom of a 150 mm petri dish with parafilm and
tape it to the bottom to keep the surface flat (Fig. 2a).
7. Transfer 23 μL of the hybridization solution from step 5 onto
the parafilm, one drop for each coverslip. By using the forceps,
take the coverslip from each well, remove from each coverslip
the leftover pre-hybridization solution using a Kimtech tissue,
and place each coverslip face down onto the prepared hybridization drop. Place a small container (e.g., the cap of a 15 mL
plastic tube) toward the edge of the Petri dish and fill it with
DDW. Cover and seal the petri dish with parafilm to create a
hybridization chamber. Incubate in the dark at 37
C for 3 h.
Incubate at 37
C the pre-hybridization solution that you will
use for the following washes (step 8).
58
Evelina Tutucci and Robert H. Singer