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Juvenile No Acid Bone & Cartilage Stain

Juvenile No Acid Bone & Cartilage Stain

Jan 31, 2011

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