Juvenile No Acid Bone & Cartilage Stain

Optimized for 21 dpf zebrafish juveniles but it works well on other similarly sized fish.

Protocol

Anesthetize fish with MESAB (tricaine) in tubes, fill to top with no airspace and capped, lay tubes on their sides until all gill movement stops.
Fix: 2% PFA 3 hrs @ room temperature and gently rock on horizontal rotator.
Wash: Tris/MgCl₂30 min or more on rotator
Stain: 0.02% Alcian Blue/10mM MgCl2, pH 7.5; overnight @ room temperature on rotator
Differentiate/ Re-hydrate:

80% EtOH/100mM Tris, pH7.5/10mM MgCl₂; 30 min or more on rotator

50% EtOH/100mM Tris, pH7.5; 30 min or more on rotator

25% EtOH/100mM Tris, pH7.5; 30 min or more on rotator

0.5% KOH; 30 min or more on rotator

Bleach: 3% H₂O₂/ 0.1% KOH 30 minutes exact; uncapped, upright tubes
Rinse: 35% Sat. NaBO₄; 30 min or more on rotator
Clear/digest: 1% Trypsin in 35% Saturated NaBO₄ 30 minutes; on horizontal rotator, gently

Wash: 10% glycerol/0.5% KOH; 30 minutes or more, on rotator, gently
Stain: 0.01% Alizarin, pH 7.5, overnight @ room temperature, on rotator, gently
Differentiate: 50% glycerol/0.1% KOH; 1 quick wash, 30 min or more on rotator, gently;
2nd wash without agitation overnight or more@ room temperature.
Store: without agitation (at 4C if possible)

Long term storage: 70%-100% glycerol (in H₂O, not KOH) with a couple of thymol crystals; at room temperature.

NOTE THAT SOME SOLUTIONS ARE DIFFERENT THAN FOR 5-7dpf FISH

SOLUTIONS

2%PFA/1XPBS pH7.5 for 50ml:

12.5ml 8% PFA

5ml 10XPBS

q.s. to 50ml water

10mM 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.

0.02% Alcian/25mM MgCl₂ Stain pH 7.5

To make 50 mls:

5ml 0.2% Alcian blue (Anatech, Ltd. Cat. # 862) in 90%ETOH

5ml 1M Tris pH 7.5

1.25ml 1M MgCl₂

32.6ml 95% ETOH

6.15ml water

Ethanol rehydration:

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 pH7.5

18.7ml water

25% ETOH:

13.2ml 95% ETOH

5ml 1M Tris pH 7.5

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.

0.01% Alizarin Stain/10% glycerol/0.5%KOH

To make 50 mls:

1ml 0.5% Alizarin Stock

5ml 100% glycerol

1.25ml 20%KOH

42.75ml water

10% glycerol/0.5%KOH

To make 50mls:

5ml 100% glycerol

1.25ml 20% KOH

43.75ml water

50% glycerol/0.1% KOH

To make 50 mls:

25ml 100% glycerol

250μl 20% KOH

23.75ml water

35% saturated sodium borate

To make 500mls: 8.3 g sodium borate decahydrate

Q.S. to 500ml

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/35% saturated sodium borate

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

3% H₂0₂/0.1%KOH:

Make ahead of time and keep at 4C. Bring to room temperature before using to reduce bubbles trapped in fish.

For 50mls:

5ml 30%H202

250μl 20%KOH

44.75ml water

MESAB

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 the right pH 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 solution clears. Q.S. to 100 ml.

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 & Histochemistry 2007, 82(1): 23-28