Materials and reagents

Biological materials

1. Zebrafish (Danio rerio, Riken Wild-type (RW) strain, the National BioResource Project Zebrafish in Japan)

2. Medaka (Oryzias latipes, Hd-rR strain, the National BioResource Project Medaka in Japan)

1. Calcein (Fujifilm Wako, catalog number: 340-00433)

2. NaOH (Fujifilm Wako, catalog number: 194-18865)

3. NaCl (Fujifilm Wako, catalog number: 191-01665)

4. KCl (Fujifilm Wako, catalog number: 160-03555)

5. CaCl₂ (Fujifilm Wako, catalog number: 038-19735)

6. HEPES (Fujifilm Wako, catalog number: 342-01375)

7. Alizarin red S (Fujifilm Wako, catalog number: 011-01192)

8. KOH (Fujifilm Wako, catalog number: 168-21815)

9. Tricaine (Fujifilm Wako, catalog number: 051-06571)

10. 2-Phenoxyethanol (Fujifilm Wako, catalog number: 163-12075)

11. Methylcellulose 1500 cP (Fujifilm Wako, catalog number: 139-02145)

1. 2.0% calcein stock solution (see Recipes)

2. 0.2% calcein staining solution (see Recipes)

3. 1× Ringer’s solution (see Recipes)

4. 1/3× Ringer’s solution (see Recipes)

5. 0.1% alizarin red S stock solution (see Recipes)

6. 0.01% alizarin red S staining solution (see Recipes)

7. Tricaine (MS-222) anesthetizing stock solution for zebrafish (see Recipes)

8. 2.0% methylcellulose solution (see Recipes)

1. 2.0% calcein stock solution

   Reagent	Final concentration	Amount
   Calcein	2.0%	1 g
   0.5 N NaOH solution	Adjust to pH 7.0
   1/3 Ringer’s solution (Recipe 4)	n/a	Add up to 50 mL
   Total	n/a	50 mL

   The prepared 2.0% calcein stock solution should be aliquoted into small tubes and stored at -20 °C in the dark. Instead of 1/3 Ringer’s solution, E3 or similar buffers can be used. Prepare a 0.5 N NaOH solution by dissolving 1.0 g of NaOH in 50 mL of distilled water.




2. 0.2% calcein staining solution

   Reagent	Final concentration	Amount
   2.0% calcein stock solution (Recipe 1)	0.2%	300 μL
   1/3 Ringer’s solution (Recipe 4)	n/a	2.7 mL
   Total	n/a	3.0 mL

   0.2% calcein staining solution should be prepared prior to staining. The example shown above is for a 35 mm dish; the volume to be added is 3.0 mL. If more dishes are used, the volume can be increased. Instead of 1/3 Ringer’s solution, E3 or similar buffers can be used.




3. 1× Ringer’s solution

   Reagent	Final concentration	Amount
   NaCl	116 mM	6.78 g
   KCl	2.9 mM	0.216 g
   CaCl2·2H2O	1.8 mM	0.265 g
   HEPES	5.0 mM	1.192 g
   0.5 N NaOH solution	Adjust to pH 7.0
   DW	n/a	Add up to 1,000 mL
   Total	n/a	1,000 mL

   1× Ringer’s solution is stable at room temperature for years.




4. 1/3× Ringer’s solution

   Reagent	Final concentration	Amount
   1× Ringer’s solution (Recipe 3)	1/3×	333 mL
   DW	n/a	Add up to 1,000 mL
   Total	n/a	1,000 mL

   1/3× Ringer’s solution is stable at room temperature for years.




5. 0.1% alizarin red S stock solution

   Reagent	Final concentration	Amount
   Alizarin red S	0.1%	0.05 g
   1 N KOH solution	Adjust to pH 7.4
   1/3 Ringer’s solution (Recipe 4)	n/a	Add up to 50 mL
   Total	n/a	50 mL

   0.1% alizarin red S stock solution should be stored at room temperature in the dark. Instead of 1/3 Ringer’s solution, E3 or similar buffers can be used. Prepare a 1 N KOH solution by dissolving 2.0 g of KOH in 50 mL of distilled water.




6. 0.01% alizarin red S staining solution

   Reagent	Final concentration	Amount
   0.1% alizarin red S stock solution (Recipe 5)	0.01%	300 μL
   1/3 Ringer’s solution (Recipe 4), or DW	n/a	2.7 mL
   Total	n/a	3.0 mL

   0.01% alizarin red S staining solution should be prepared prior to staining. The example shown above is for a 35 mm dish; the volume to be added is 3.0 mL. If more dishes are used, the volume can be increased. Instead of 1/3 Ringer’s solution, E3 or similar buffers can be used.




7. Tricaine (MS-222) anesthetizing stock solution for zebrafish

   Reagent	Final concentration	Amount
   Tricaine	2.0%	1 g
   DW		Add up to 50 mL
   Total	n/a	50 mL

   The prepared tricaine anesthetizing stock solution should be divided into small tubes and stored at -20 °C in the dark.




8. 2.0% Methylcellulose solution

   Reagent	Final concentration	Amount
   Methylcellulose 1500 cP	2.0%	1 g
   DW	n/a	Add up to 50 mL
   Total	n/a	50 mL

   To completely dissolve the methylcellulose in DW, shake vigorously for several days. When photographing, the fish is placed in this viscous solution to orient the fish in the desired position. It is possible to adjust the solution to a percentage of 2%–3% methylcellulose depending on personal preference.

Laboratory supplies

1. Non-treated dish, 35 mm (IWAKI, catalog number: 1000-035)

2. Glass-base dish, 35 mm (IWAKI, catalog number: 3970-035)

3. Transfer pipette, 3 mL (BD Falcon, catalog number: 357575)

Equipment

1. Fluorescent stereomicroscope (Leica, model: M205 FA)

2. Digital camera (Leica, model: DFC350 FX)

Software and datasets

1. ImageJ (v. 1.53e, September 2020, free to use, https://imagej.net/ij/)

Procedure