RNA Detection

(a) In the KNIME.ini file in your KNIME installation folder:

set Xmx to –Xmx4g or –Xmx6g and –XX:MaxPermSize to

–XX:MaxPermSize¼512 m.

(b) Ensure your KNIME update sites include “KNIME Ana-

lytics Platform Update Site” and “Stable Community

Contributions”.

(c) Add update site “MPI-CBG” with location ‘https://com

munity.knime.org/download/de.mpicbg.knime.ip.

update’

(d) Install the following tools (seeNote 7):

l KNIME External Tool Support (Labs).

l KNIME Image Processing.

l KNIME Image Processing—Supervised Image

Segmentation.

l KNIME Quick Forms.

l KNIME Quick Forms (legacy).

l KNIME Virtual Nodes.

(e) Install the KNIME MS-ECS-2D workflow via “tinyurl.

com/KNIME-MS-ECS”. On this page you can also find

more background information on how to install KNIME.

(f) The pipeline is available in versions for Mac or Linux.

3 Methods

Carry out all washes and rinses in 1 mL unless otherwise specified.

Use RNase-free solutions and wear gloves at all times to pre-

vent RNase contamination.

3.1 Embryo
Embedding

1. Collect embryos in the chorion in a petri dish and let them
   develop in embryo medium to the desired stage (seeNote 8).


2. Fix embryos in a round bottom 2 mL tube in 4% formaldehyde
   in PBT overnight at 4C or for 4 h at RT.


3. Rinse embryos in PBTand manually dechorionate them in PBT
   under a dissecting scope using sharp forceps. Put embryos back
   in a 2 mL tube with PBT.


4. Equilibrate embryos in 30% sucrose in PBS until they sink (see
   Note 9).


5. Rinse embryos twice in fresh 30% sucrose in PBS (seeNote 10).


6. Replace with fresh 30% sucrose in cryo incubation solution.
   Make sure that embryos are mixed well with the medium and
   leave for 5 days at 4C(seeNote 11).


7. Equilibrate embryos in OCT by moving them through two
   consecutive baths of OCT (seeNotes 12and 13 ) (Fig.2a).

smFISH and Automated Analysis in Zebrafish 149