conditions to your aliquot of proteinase K since excessive pro-
tein digestion will affect sample integrity.- If signal/noise is poor in your samples, the best way to opti-
mize this is by changing probe concentrations in the hybridiza-
tion buffer. We usually test ranges from 0.2–1.5μL probe
(25μM stock solution) per 100μL buffer. - smFISH can easily be combined with antibody staining to, for
example, boost the signal of a transgenic line, or to detect the
protein encoded by your mRNA of interest. For antibody
staining, add the primary antibody to the overnight hybridiza-
tion step (Subheading3.4,step 9) and the secondary antibody
to the first wash step (Subheading3.4,step 12). Antibody
staining can also be performed after smFISH staining. Be
aware that this will lead to a reduction of smFISH signal. - GLOX mounting medium is a simple and quick mounting
medium which can be used when imaging within 24 h of
sample preparation (coverslips can be stored in GLOX buffer
at 4C until mounting and imaging). For longer-term storage,
other mounting mediums like prolong GOLD or Vectashield
are recommended. When using mounting mediums like pro-
long GOLD or Vectashield, make sure that no liquid is left on
the sample before mounting. A small amount of liquid can
form a barrier between the sample and the mounting medium,
preventing the mounting medium from penetrating the sam-
ple. This will lead to rapid bleaching of your sample. - Acquire images starting with the longest wavelength and work
your way down to shorter wavelengths. This will minimize
bleaching across fluorophores because shorter wavelengths
carry higher energy levels and induce more bleaching. - Using z-spacing of maximally 0.3μm ensures that individual
transcripts are detected in more than one z-slice and that no
transcripts are missed in the acquisition. This aids computa-
tional transcript detection. - Pressing the letter “l” in Fiji will open a command searching
window. Although all commands are accessible through the
menu, this is a quick way to find commands without having to
browse through the menu structure. - The KNIME workflow consists of a training phase and a pre-
diction phase (Fig.4). After training the workflow once, it can
be used to produce predictions for different samples. We have
trained the pipeline on zebrafish membrane images, and have
produced high-quality predictions for samples from zebrafish
as well as other species using this pipeline. Therefore, we
suggest that you use our pretrained pipeline on your samples.
If the available pipeline produces poor results for your samples,
you can consider retraining the pipeline with your own
160 L. Carine Stapel et al.