- Glass pipet.
- 6-well staining plate (Fig.2a).
- Embryo manipulator (e.g., nickel-plated pin holder with
curved pin, Fine Science Tools). - Large liquid nitrogen container (Fig.2c).
- Large sieve (Fig.2c).
- 250 mL beaker.
- Sharp forceps.
- Blunt-end forceps.
- Low-temperature thermometer (to 100 C).
- Incubator set to 28C.
- Stereomicroscope.
2.2 Cryosectioning 1. Glass cleaning detergent (e.g., Mucasol).
- Milli-Q water.
- 100% ethanol.
- 0.1 w/v % poly-L-lysine (MW 150,000–300,000 Da) in Milli-
Q water. - 2222 mm selected #1.5 coverslips (0.17+/0.0005 mm)
(seeNote 2). - Coverslip holder (e.g., XL Wash-N-Dry coverslip rack, Diver-
sified Biotech) (Fig.3b). - Slide staining dish or beaker to fit coverslip holder (Fig.3b).
- Anti-roll plate for cryostat.
- Cryostat blade.
- Specimen stage for cryostat.
- Thick and thin brush.
- 6-well plate.
- Parafilm.
- Sonication bath.
- Cryostat (e.g., Microm HM560).
2.3 smFISH (Incl.
Imaging)
- 70% ice-cold ethanol in Milli-Q water (store at 20 C).
- 2SSC diluted from 20SSC (commercial, RNase free).
- 5μg/mL proteinase K in 2SSC.
- smFISH wash buffer: 10% formamide and 2SSC in nuclease-
free water (seeNote 3). - smFISHhybridizationbuffer: 10w/v%dextransulfate,10v/v%
formamide, 1 mg/mL E. coli tRNA, 2SSC, 0.02 w/v % BSA,
and 2 mM Vanadyl-ribonucleoside complex in nuclease-free
water (seeNote 3).
smFISH and Automated Analysis in Zebrafish 147