blotting off the excess wash buffer, do not let the cells dry out
as this will damage them.
8. After 20 s, remove the coverslip and place it cell side up into
one of the free wells of the 12-well plate with 4% paraformaldehyde (see step 4). At this stage the cells are fixed and can be
left aside until all the other coverslips are processed.
9. Repeat steps 6–8 for the remaining coverslips. In addition to
the main experiment, a series of control experiments should
ideally be performed in tandem to validate the smFISH data
(see Notes 9, 14–17). Other experiments can be conducted
to ensure that the cytosol has been completely extracted (see
Note 18).
10. Let coverslips sit in fixing solution for at least 15 min at room
temperature.
11. Wash coverslips in PBS three times to remove the
paraformaldehyde.
12. After the washes, transfer the coverslips onto a porcelain coverslip staining rack, ensuring that they are fully immersed in
ice-cold methanol (À20
C) in a 250 mL beaker (see Note 6),
for 30 min. Typically, we keep the beaker in a À20
C freezer
during the course of the experiment.
13. Transfer the rack from the methanol to a new 250 mL beaker
containing room-temperature PBS (about 200 mL) to rehydrate the cells for 15 min.
14. Transfer the rack to a new 250 mL beaker with roomtemperature PBS (about 200 mL) a second time to remove
any remaining methanol and allow to sit for 15 min.
3.2 smFISH Staining
and Mounting onto
Slides
1. To prepare for the next washing steps, lay down a strip of
parafilm on a wet flat surface, and remove any excess water,
bubbles, or creases using RNase-free gloves.
2. For each coverslip, pipette 500 μL of 10% formamide wash
buffer (see Subheading 2.3, item 3, Note 19) onto the parafilm, such that it forms a firm liquid droplet.
3. Pick up each coverslip with forceps and quickly wipe the
non-cell side with a Kimwipe until it is dry (see Note 20).
Carefully transfer each coverslip cell side down onto the droplet
of 10% formamide wash buffer and incubate for 5 min.
4. Repeat Subheadings 3.1, steps 1–3, to wash a second time.
5. Pipette 100 μL of FISH hybridization buffer with diluted
Stellaris probes (see Subheading 2.3, item 4), or scrambled
probes as control (see Note 21), onto the parafilm in the
smFISH staining dish (see Subheading 2.3, item 5), so that it
42
Jingze J. Wu and Alexander F. Palazzo
as this will damage them.
8. After 20 s, remove the coverslip and place it cell side up into
one of the free wells of the 12-well plate with 4% paraformaldehyde (see step 4). At this stage the cells are fixed and can be
left aside until all the other coverslips are processed.
9. Repeat steps 6–8 for the remaining coverslips. In addition to
the main experiment, a series of control experiments should
ideally be performed in tandem to validate the smFISH data
(see Notes 9, 14–17). Other experiments can be conducted
to ensure that the cytosol has been completely extracted (see
Note 18).
10. Let coverslips sit in fixing solution for at least 15 min at room
temperature.
11. Wash coverslips in PBS three times to remove the
paraformaldehyde.
12. After the washes, transfer the coverslips onto a porcelain coverslip staining rack, ensuring that they are fully immersed in
ice-cold methanol (À20
C) in a 250 mL beaker (see Note 6),
for 30 min. Typically, we keep the beaker in a À20
C freezer
during the course of the experiment.
13. Transfer the rack from the methanol to a new 250 mL beaker
containing room-temperature PBS (about 200 mL) to rehydrate the cells for 15 min.
14. Transfer the rack to a new 250 mL beaker with roomtemperature PBS (about 200 mL) a second time to remove
any remaining methanol and allow to sit for 15 min.
3.2 smFISH Staining
and Mounting onto
Slides
1. To prepare for the next washing steps, lay down a strip of
parafilm on a wet flat surface, and remove any excess water,
bubbles, or creases using RNase-free gloves.
2. For each coverslip, pipette 500 μL of 10% formamide wash
buffer (see Subheading 2.3, item 3, Note 19) onto the parafilm, such that it forms a firm liquid droplet.
3. Pick up each coverslip with forceps and quickly wipe the
non-cell side with a Kimwipe until it is dry (see Note 20).
Carefully transfer each coverslip cell side down onto the droplet
of 10% formamide wash buffer and incubate for 5 min.
4. Repeat Subheadings 3.1, steps 1–3, to wash a second time.
5. Pipette 100 μL of FISH hybridization buffer with diluted
Stellaris probes (see Subheading 2.3, item 4), or scrambled
probes as control (see Note 21), onto the parafilm in the
smFISH staining dish (see Subheading 2.3, item 5), so that it
42
Jingze J. Wu and Alexander F. Palazzo