- PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 ,
1.47 mM KH2PO4 in nuclease-free water. Adjust pH to 7.4
with HCl. - PBT: 0.1 v/v % Tween 20 in PBS.
- Fixative: 4% formaldehyde in PBT.
- Cryoprotection solution: 30% sucrose in PBS.
- Tissue-Tek®OCT compound (optimal cutting temperature
compound) (Fig.2a). - Cryo incubation solution: prepare a solution of 30% sucrose in
a 50/50 mixture of PBS with OCT. - Isopentane.
- Petri dish, 100 mm diameter.
- Caps of Eppendorf tubes (seeNote 1).
- Pipet pump for glass pipets to transfer dechorionated embryos
(e.g., The Pipet Pump, Bel-Art Scienceware) (Fig.2a).
Fig. 2Embedding of zebrafish embryos for cryosectioning. (a) Samples are equilibrated in OCT in a 6-well
staining dish and embedded in the cap of an Eppendorf tube under a dissecting scope. (b) Embryos are
oriented laterally with their animal caps facing in the same direction to ensure optimal section quality. The red
arrow on the Eppendorf cap indicates their orientation so that the cryo-block can be mounted on the cryostat
with the animal caps facing toward the blade. (c) A beaker with isopentane is cooled to 80 C in a bucket
with liquid nitrogen. A sieve may be used to hold the beaker. (d) The cryo-block is rapidly frozen in the
precooled isopentane
146 L. Carine Stapel et al.