blade temperatures of around 20 C for many other sample
types on the same cryostat.
- After mounting the cryo-block to the specimen stage, remove
the mold and check whether any cracks are present. If so, repair
cracks with OCT (use the quick-freeze option on your cryostat
to rapidly freeze the newly applied OCT). Cracks can occur due
to rapid freezing of the OCT in a confined space. Although
cracks can be prevented by freezing at lower temperatures, this
also decreases embryo integrity and is therefore not
recommended.
- The yolk can fracture during sectioning. By sectioning through
the yolk last, you ensure that this does not affect embryo
integrity.
- Sectioning results are best when the cryo-block is relatively
warm. If the block is too cold and your sections contain cracks
or roll up tightly it can help to briefly warm up the block before
cutting a section by pressing your thumb to the block for a
couple of seconds. Make sure to wear clean gloves to not
contaminate the sample with RNases!
- The anti-roll plate will keep the section flat. Lift the plate to
mount your section to a coverslip. You can use a thin brush to
prevent the section from rolling up (Fig.3c).
- We use 6–12 coverslips per embryo and put sections obtained
from multiple positions in the embryo on each single coverslip
(seeFig. 2e). Make sure to not leave the sections out at RT for
more than 20 min before storing them at 80 C as this can
affect smFISH quality.
- We store sections in 6-well plates, sealed with Parafilm. Sec-
tions can be stored at 80 C for a long time. Up to 6 months,
we have not observed a decrease in sample quality.
- Sections should be kept at 80 C for at least 1 h before
continuing with the smFISH protocol. This will improve
smFISH results.
- Add the fixative to each section immediately after taking it out
of the freezer, without letting it thaw. This will prevent RNA
degradation.
- Add solutions to the side of the well and not directly on the
coverslip to avoid dislodging the sections.
- In some protocols, sections are kept in ethanol for up to a
month. However, in our hands this reduces smFISH quality.
- This treatment is optional but improves signal/noise for most
of our probe sets. Mild proteinase K treatment can digest
proteins that cover the RNA of interest and reveal smFISH
probe binding sites. Make sure to optimize treatment
smFISH and Automated Analysis in Zebrafish 159
