forms a firm liquid droplet. Pipette as many droplets as there
are coverslips to be stained.
6. Pick up each coverslip with forceps and quickly wipe the
non-cell side with a Kimwipe until it is dry and free of salt
crystals (see Note 20). Carefully transfer each coverslip cell
side down onto the droplet of FISH hybridization buffer
with diluted Stellaris probes, so that the coverslip floats on
top of the liquid, leaving its cell side fully immersed while
leaving the opposite side dry.
7. After transferring all the coverslips onto the parafilm, place the
lid onto the petri dish and seal edges with parafilm. Place the
petri dish into a 37
C cell incubator for 24–48 h.
8. Again, lay down a fresh strip of parafilm on a wet flat surface,
and remove any excess water, bubbles, or creases using RNasefree gloves.
9. For each coverslip pipette three droplets of 500 μL of 10%
formamide wash buffer onto the parafilm.
10. Remove the petri dish from the 37
C cell incubator. Slowly
pipette 200 μL of 10% formamide wash buffer directly beside
each coverslip and allow for the wash buffer to seep beneath
each coverslip, floating it above the parafilm (see Note 22).
11. Carefully pick up the coverslip with forceps, transfer it onto one
of the wash droplets on the strip of parafilm (see Subheading
3.2, step 9), and let sit for 5 min at room temperature. Repeat
this process twice more by transferring each coverslip onto a
fresh droplet of wash buffer after 5 min.
12. The smFISH-stained cells can at this stage be immunostained
to co-localize smFISH foci with ER markers (see Note 23).
13. In between washes, prepare sterile glass slides by pipetting
~15 μL of DAPI mounting solution onto each glass slide (see
Note 11).
14. After three washes, carefully remove each coverslip from the
droplets of wash buffer and dry any excess liquid with Kimwipes. Each coverslip is then placed, cell side down, onto a
droplet of DAPI mounting solution on the glass slides.
15. Slides are then labeled and can be stored at 4
C. Note that
slides should be allowed to dry for at least 24 h prior to
imaging.
3.3 Imaging
and Quantification
1. smFISH-stained cells are imaged with an epifluorescence or a
confocal microscope using a 60Â PlanApo objective (see
Note 24).
2. Acquire images of the smFISH and DAPI channel for each field
(see Note 25).
Visualizing ER-Associated mRNA
43
are coverslips to be stained.
6. Pick up each coverslip with forceps and quickly wipe the
non-cell side with a Kimwipe until it is dry and free of salt
crystals (see Note 20). Carefully transfer each coverslip cell
side down onto the droplet of FISH hybridization buffer
with diluted Stellaris probes, so that the coverslip floats on
top of the liquid, leaving its cell side fully immersed while
leaving the opposite side dry.
7. After transferring all the coverslips onto the parafilm, place the
lid onto the petri dish and seal edges with parafilm. Place the
petri dish into a 37
C cell incubator for 24–48 h.
8. Again, lay down a fresh strip of parafilm on a wet flat surface,
and remove any excess water, bubbles, or creases using RNasefree gloves.
9. For each coverslip pipette three droplets of 500 μL of 10%
formamide wash buffer onto the parafilm.
10. Remove the petri dish from the 37
C cell incubator. Slowly
pipette 200 μL of 10% formamide wash buffer directly beside
each coverslip and allow for the wash buffer to seep beneath
each coverslip, floating it above the parafilm (see Note 22).
11. Carefully pick up the coverslip with forceps, transfer it onto one
of the wash droplets on the strip of parafilm (see Subheading
3.2, step 9), and let sit for 5 min at room temperature. Repeat
this process twice more by transferring each coverslip onto a
fresh droplet of wash buffer after 5 min.
12. The smFISH-stained cells can at this stage be immunostained
to co-localize smFISH foci with ER markers (see Note 23).
13. In between washes, prepare sterile glass slides by pipetting
~15 μL of DAPI mounting solution onto each glass slide (see
Note 11).
14. After three washes, carefully remove each coverslip from the
droplets of wash buffer and dry any excess liquid with Kimwipes. Each coverslip is then placed, cell side down, onto a
droplet of DAPI mounting solution on the glass slides.
15. Slides are then labeled and can be stored at 4
C. Note that
slides should be allowed to dry for at least 24 h prior to
imaging.
3.3 Imaging
and Quantification
1. smFISH-stained cells are imaged with an epifluorescence or a
confocal microscope using a 60Â PlanApo objective (see
Note 24).
2. Acquire images of the smFISH and DAPI channel for each field
(see Note 25).
Visualizing ER-Associated mRNA
43