3.4 smFISH 1. Take coverslips with sections from^80 C and place them in a
6-well plate (Fig.3e). Post-fix the sections in 4% formaldehyde
in PBS for 15 min (seeNotes 26and 27 ).
- Rinse sections twice with PBS.
- Rinse with 70% ice cold ethanol and replace with fresh 70%
ethanol. Keep for 4–8 h at 4C to permeabilize the sample (see
Note 28). - Rehydrate the sections in 2SSC.
- Treat with 5μg/mL proteinase K in 2SSC for 10 min while
gently shaking (seeNote 29). - Wash 25 min with 2 mL 2SSC while gently shaking.
- Rinse sections with wash buffer and replace with fresh wash
buffer. Let equilibrate for 5 min. - Thaw 100μL hybridization buffer per sample and add 0.7μL
smFISH probe (25μM) (seeNotes 4, 30and 31 ). - Coat the bottom of a petri dish with Parafilm and place a 95μL
drop of hybridization buffer with probe on the Parafilm
(Fig.3f). Take a coverslip with sample and carefully remove as
much wash buffer as possible with a piece of filter paper with-
out touching the sections. Carefully place the coverslip section-
down on the drop of hybridization buffer (Fig.3f), close but
do not seal the petri dish and incubate overnight for 14–16 h at
30 C. - The next day, pipet 100μL wash buffer on the corner of the
coverslip and carefully peel the coverslip off the Parafilm,
making sure to not dislodge the sample. Place the coverslip in
a 6-well plate with the sections facing up. - Rinse once with wash buffer.
- Wash twice with wash buffer for 30 min at 30C without
shaking. For membrane staining, add 1:100 phalloidin–Alexa
488 (or another fluorophore) to the second wash step (see
Note 5). For DNA staining, add 0.5μg/mL DAPI to the
second wash step. - Rinse once with GLOX buffer and leave in fresh GLOX buffer
at 4C until mounting (seeNote 32). - Mount in GLOX mounting medium. Place 25μL GLOX
mounting medium on a microscope slide and slowly lower
the smFISH sample down on the drop to prevent bubbles.
Remove excess mounting medium by carefully touching a
piece of filter paper to the side of the coverslip. - Tightly seal the sample with nail polish to prevent evaporation
of the mounting medium and proceed to imaging.
smFISH and Automated Analysis in Zebrafish 151