- Custom Stellaris smFISH probes, diluted to 25μM in Milli-Q
water (seeNote 4). - 1 mg/mL DAPI.
- Phalloidin–Alexa 488 (seeNote 5).
- GLOX buffer: 10 mM Tris, pH 7.5, 2SSC, and 0.4 w/v %
glucose (in nuclease-free water). - GLOX mounting medium: GLOX buffer, 47μg/mL glucose
oxidase, and 0.58 mg/mL catalase suspension. - Microscope slides.
- Nail polish.
- Epifluorescence microscope with suitable filter sets, a high NA
objective, and a sensitive camera (seeNote 6).
2.4 Data Analysis For additional information on how to install the plugins, you may
consult the documentation associated with Stapel et al. 2016 [15].
- Fiji (http://fiji.sc/Fiji)
(a) Activate update sites “MS-ECS-2D” and “3D ImageJ
Suite”. - KNIME
Background information and more details on how to install
KNIME for the applications described in this chapter can be
found athttp://tinyurl.com/KNIME-MS-ECS. Briefly:
Fig. 3Sample preparation for smFISH.(a) The cryo-block is mounted on the cryostat with the animal caps
facing toward the blade of the cryostat (red arrow). (b) Coverslips are placed in a coverslip holder and are
coated with poly-L-lysine in a slide staining dish to increase adhesion of sections to the coverslips. (c) Multiple
sections can be placed on a single coverslip. (d) Cryosections on a coverslip. Note the orientation. While the
yolk is damaged, the animal caps are intact. (e) Coverslips with sections are placed section-up in a 6-well
plate for smFISH washes. (f) Coverslips are placed section-down on a drop of hybridization mix in a Parafilm-
coated petri dish for probe hybridization overnight
148 L. Carine Stapel et al.