have also obtained good results with a 601.3NA silicon
objective. It is also possible to image your samples on a
spinning disk microscope with comparable objectives and cam-
era, but results might be more difficult to interpret for probe
sets with low signal to noise ratios.- Uncheck “Group items by category” to be able to search
through an alphabetically ordered list of all tools. This makes
it easy to find the required tools. - While we optimized the protocol for embryonic stages up to
gastrulation (shield stage), it also works well at later stages. We
will indicate small changes in the embedding procedure that
can be taken into account when working with post-gastrulation
stages. - Embryos at pregastrulation stages will sink within 30 min; later
stages might take longer to sink. - It is important to rinse the embryos several times in sucrose/
PBS to remove any Tween remaining from the fixation step, as
this decreases embedding quality. - While a 5-day incubation time seems excessive, in our hands it
has led to greatly improved section quality at (pre)gastrulation
stages without loss of RNA signal. Make sure to keep embryos
in the dark if you are working with a fluorescent transgenic line
(seeNote 5). At post-gastrulation stages, 5-day incubation is
not necessary and one can proceed with the next steps of the
protocol as soon as embryos sink to the bottom of the tube. - We prefer using 6-well staining plates and an embryo manipu-
lator (Fig.2) to move embryos through OCT (seeSubheading
2.1,items 14and 15 ; Fig.2a). - From this step onward keep embryos and liquids at 4Cas
much as possible to enhance freezing speed and improve
embryo integrity. - Make sure to orient all embryos in the same direction and note
the orientation on the embedding cap/mold with a marker.
This will improve section quality later (seeNote 20) (Fig.2b). - Check the temperature with a low-temperature thermometer.
After use, isopentane can be stored in a glass bottle and reused. - Section quality will be optimal if blocks are sectioned within 1
week. However, it is possible to obtain good sections when
blocks have been stored for several months. - Poly-L-lysine for coverslip coating can be stored in a plastic
container and reused up to three times. - Temperatures might need to be optimized depending on your
cryostat. However, we recommend to use relatively high tem-
peratures for the best results. As a reference, we use block and
158 L. Carine Stapel et al.