Recipes/Staining

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KIMMEL LAB STANDARD DOUBLE BONE/CARTILAGE STAIN PROTOCOL from work by Macie Walker for Walker & Kimmel, Informa '06

for zebrafish embryos, 4-6 dpf:

1) MESAB embryos, fill tubes to the top with MESAB solution and lay on tubes on their sides for about 5 minutes; remove most of liquid.

2) Fix: Add 2%PFA/1X PBS. Nutate 1 hr, with tubes on their sides. Warning: Longer fixing may decrease bone staining.

3) Wash 1x10 mins with 100mM Tris pH7.5/10mM MgCl2.

4) Alcian stain: Add 1 ml of 0.04% Alcian/10mM MgCl2 stain pH 7.5. Nutate overnight. Warning: Embryos tend to get stuck in bottom of tube. Check for this more than once during staining. Wash with 80% ETOH/100mM Tris pH7.5/10mM MgCl2 for 5 mins. Wash with 50% ETOH/100mM Tris pH7.5 for 5 mins. Wash with 25% ETOH/100mM Tris pH7.5 for 5 mins.

5) Bleach: Add 1ml 3%H202/0.5%KOH. Let tubes sit with lids open, for 10 mins. Warning: Bleach can reduce bone staining.

6) Wash: 2x10 mins with 1 ml 25% glycerol/0.1% KOH. Rinse bleach out well. If bubbles are present, then bleach is not rinsed out enough.

7) Alizarin stain: Add 1 ml of 0.01% Alizarin stain, pH 7.5. Nutate 30 mins.

8) Destain: a. Add 1 ml 50% glycerol/0.1%KOH. b. Nutate 10 mins, then replace with fresh 50% glycerol/0.1%KOH.

Warning: Some bone staining may fade if left on nutator overnight. If not viewing within a few hours, take embryos off nutator and store in rack, without agitation.