tissue quickly without promoting crystallization even at the
most extreme temperatures.- Wash buffer (1PBT): To make 1 L of PBT, add 1 mL of
Triton-X-100 to 1 L of PBS (see Note 2). Store at room
temperature indefinitely. - Red detection reagent: For the detection of alkaline phospha-
tase (AP), solutions AP-Fast Red B and A of the RNAScope kit
must be combined in a 1:60 ratio in a separate centrifuge tube
to create a working solution (seeNote 2). - Green detection reagent: To detect horseradish peroxidase
(HRP, make a 1:50 ratio mixture of HRP-Green-B to HRP-
Green-A of the RNAScope kit in a separate centrifuge tube (see
Note 3). - EcoMount mounting medium (Biocare).
2.2 Equipment 1. Cryostat equipped with a microtome blade (e.g., Leica
1860UV cryostat).
- Plastic embedding molds.
- Iridectomy scissors.
- Dumont #5 fine-tip forceps.
- 1.5 mL Eppendorf tubes.
- Superfrost/Plus microscope slides (e.g., Fisherbrand).
- Drierite desiccant.
- Coplin jars.
- 500 mL beaker.
- Aluminum foil.
- Thermometer.
- Heating plate.
- Hydrophobic pen.
- Paper towels.
- Modified Tupperware (seeFig. 2 andNote 4).
- Serological pipettes.
- Hybridization incubator.
3 Methods
3.1 Tissue
Preparation
The zebrafish heart is a two-chambered organ with one atrium and
ventricle (Fig.3). For orientation purposes during histology, it is
important to extract the heart with the chambers and the outflow
tract together. All methods are carried out at room temperature
unless otherwise specified.200 Viravuth P. Yin