4 Notes
- We use Eppendorf caps to embed our samples. Alternatively,
commercial molds can be used but we prefer the small size of
Eppendorf caps. For early stages, coloring the bottom of the
cap/embedding mold with a black marker can improve visibil-
ity of the embryos (Fig.2b). - Mounting sections on coverslips instead of slides improves
light transmission and image quality because it places the sam-
ple directly at the microscope objective without a barrier of
mounting medium. We prefer using 2222 mm coverslips.
Although protocols from the company that sells Stellaris
smFISH probes suggest to use #1 coverslips, we obtain optimal
results with #1.5 coverslips. Selected #1.5 coverslips are opti-
mized for the light path of most microscopes. - The concentration of formamide in the smFISH and hybridi-
zation buffers can be increased from 10% to up to 25% to
reduce background signal. However, we find that this often
results in a loss of signal and find that optimizing probe con-
centration is a more efficient way to increase the signal to noise
ratio (seeNote 28). - Probes can be designed using freely available software from
Stellaris (https://www.biosearchtech.com/support/tools/design-soft
ware/stellaris-probe-designer). We recommend blasting the
probe sequences that are suggested by this webtool against
the zebrafish genome to make sure they exclusively hybridize
to your transcript of interest. When choosing a fluorophore for
probe labeling, select fluorophores in the red or far-red spec-
trum. Fluorophores with shorter wavelengths produce poor
results due to auto fluorescence in zebrafish samples. Which
exact fluorophore is optimal for your needs will depend on the
filter sets on your microscope. - We use phalloidin to mark cell outlines and enable automated
cell segmentation. Alternatively, membranes can be visualized
using a transgenic line (e.g., a lyn::fluorescent protein line), or
by injecting mRNA that codes for a membrane localized fluo-
rescent protein at the 1-cell stage. For the choice of fluorescent
protein, we have obtained good results with tdTomato, which
was still visible after the smFISH protocol. GFP, on the other
hand, loses its activity in the smFISH protocol and will need to
be detected using an antibody (seeNote 29). - We use a Delta Vision system equipped with an Olympus
UPlan SApochromat 100 1.4 oil objective, an EDGE/
sCMOS camera and the following filter sets: 435/48 (DAPI),
525/36 (Alexa 488), 594/45 (CalFluor 610-labeled smFISH
probes), 676/34 (Quasar 670-labeled smFISH probes. We
smFISH and Automated Analysis in Zebrafish 157