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For fish 40 mm Standard Length or over.


All steps except last on rotator

  • Fix: 2% PFA  overnight @ room temperature
  • Wash: Tris/MgCl2; 1 hr
  • Stain: 0.01% Alcian Blue in10mM MgCl2, pH 7.5; overnight @ room temperature
  • Differentiate/ Re-hydrate: 80% EtOH/100mM Tris, pH 7.5/10mM MgCl2; 30 min or more

50% EtOH/100mM Tris, pH 7.5; 30 min or more

25% EtOH/100mM Tris, pH 7.5; 30 min or more

0.5% KOH; 30 min or more

  • Bleach: 3% H2O2; 0.5% KOH until pigment is almost gone in body & eyes are light brown; usually several hours
  • Digest/clear: 35% Sat. NaBO4 30 min or more 1% Trypsin in 35% Sat.
  • NaBO4 depending on the size of the fish; several hours to overnight @ room temperature or @37C to speed up the process or @4C to slow down the process. Lower speed of rotator after this step.
  • Wash: 10% glycerol/0.5% KOH; 1 hr or more
  • Stain: 0.02% Alizarin, pH 7.5; overnight @ room temperature
  • Differentiate: 50% glycerol/0.5% KOH; 1 quick wash, 30 min or more;
  • 2nd wash on rotator overnight or more.
  • Store: without agitation 4C @ room temperature
  • Long term storage: 70%-100% glycerol (in H2O, not KOH) with a couple of thymol crystals; @ room temperature

Solutions for adult fish:

2%PFA/1XPBS pH7.5:

per ml:

250ul 8% PFA

100ul 10XPBS

650ul water

100mM Tris pH 7.5/25mM MgCl2

To make 50 mls:

5ml 1M Tris pH 7.5

1.25ml 1 M MgCl2

43.75ml water

0.2% Alcian Blue/90%EtOH Stock:  Alcian Blue will not readily dissolve in EtOH.  

To   make 100 mls: Add 0.2g Alcian Blue 8GX powder (Anatech, Ltd. #862) to 11.2ml 50% EtOH.  Warm and occasionally swirl.  When all is dissolved, add 95%EtOH to 100 ml. Check clarity of solution under a microscope to make sure there are no precipitates.

0.01% Alcian/25mM MgCl2 Stain pH 7.5

To make 50 mls:

2.5 ml 0.2% Alcian Blue 8GX  in 90%ETOH

5ml 1M Tris pH 7.5

1.25ml 1M MgCl2

37.6ml 95% ETOH

8.65ml water

Ethanol rehydrations:

To make 50 mls:

80% ETOH/25mM MgCl2

42.1ml 95% ETOH

5ml 1 M Tris pH 7.5

1.25ml 1M MgCl2

1.65ml water

50% ETOH:

26.3ml 95% ETOH

5ml 1M Tris pH 7.5

18.7ml water

25% ETOH:

13.2ml 95% ETOH

5ml 1M Tris pH7.5

31.8ml water

0.5% Alizarin Red S stock:  To make 50 mls: add 0.25g Alizarin Red S (JT Baker cat. #A475-03) to 50 ml water.

0.02% Alizarin Stain/10% glycerol/0.5%KOH

To make 50 mls:

2ml 0.5% alizarin red S

5ml 100% glycerol

1.25ml 20%KOH

41.75ml water

10% glycerol/0.5%KOH

To make 50mls:

5ml 100% glycerol

1.25ml 20% KOH

43.75ml water

50% glycerol/0.5% KOH

To make 50 mls:

25ml 100% glycerol

1.25ml 20% KOH

23.75ml water

35% saturated sodium borate

To make 500mls: 8.3 g sodium borate decahydrate  Q.S. to 500 ml with glass distilled water. Warm solution on hot plate with stirring until fully dissolved. This should take just a few minutes. Note: a saturated solution of sodium borate at 20°C is 23.6g/500ml water.

1% trypsin in 35% saturated sodium borate

Make fresh, using Difco trypsin 250 at 0.1g per 10mls 35% saturated sodium borate.


Tricaine: 3-amino benzoic acid ethyl ester (SIGMA Cat # A-5040 or FINQUEL, Argent Laboratories #C-FINQ-UE-5G ).

Mix in fish safe container with a stir bar:

400 mg tricaine powder

800 mg Na2HPO4 (anhydrous)

100 ml glass distilled water

Adjust to ~pH 7 with a drop at a time of 1N NaOH or 1N HCl if needed but it’s

usually right if you weigh the sodium phosphate carefully and measure the water with a

graduated cylinder.

8% PFA

8 g Pelleted PFA (Ted Pella, Inc.; cat# 18501)

90 ml dH2O

25 drops 1N NaOH

Heat at very low heat and stir just until sol'n clears. Q.S. 100 ml water.

Filter and store at 4C not more than 1 week.

Modified from:

A two-color acid-free cartilage and bone stain

for zebrafish larvae

MB Walker & CB Kimmel

Biotechnic & Histochemistry2007, 82(1): 23-28