Protocols Cleared Skeletal Preps
Alizarin Red S (bone) and Alcian Blue (cartilage) staining of cleared skeletons
modified from: Manipulating the Mouse Embryo, 2nd Edition by Hogan, Beddington, Costantini, Lacy
- -100% Ethanol (EtOH; Pharmco-AAPER Cat#111000200)
- -100% Acetone (EtOH; J.T.Baker Cat#9006-33)
- -Glycerol (Fisher Cat# BP229-4)
- -Alcian Blue stain (200 mls; 0.03%(w/v) in 80% EtOH, 20% Acetic Acid)
- Alcian blue 8GX (Sigma Cat# A5268), 0.06 g
- 100% EtOH, 160 ml
- 100% Glacial Acetic Acid, 40 ml
- -Alizarin Red stain (200 mls; 0.05% in 1% (w/v) KOH)
- Alizarin Red S (Sigma Cat#A5533), 10 mg
- Potassium Hydroxide (KOH; Sigma #P5859), 2 g
- dH2O, 200 ml
- At autopsy, place embryo in tap water for 1-5 hours (optional)
- Scald embryo in hot tap water (~65C) for 20-30 seconds for easier maceration of the tissue
- Use forceps to eviscerate the embryo.
- Fix embryo in 95% EtOH overnight. Be sure to remove all bubbles from the body cavity
- Transfer embryos to 100% Acetone and incubate overnight at room temperature
- Stain for cartilage by placing enough Alcian blue stain to cover the body. Stain overnight at room temperature.
Be sure to remove all bubbles from the body cavity
- Rinse embryos twice in 95% EtOH. Destain embryos in 95% EtOH overnight at room temperature.
- Slightly clear the sample by placing in 1% KOH for 1 hour.
- Counterstain bone in Alizarin Red stain for 3-4 hours.
- Clear the samples by placing in 1% KOH of decreasing strengths:
Initially, place embryo in 1% KOH for 1-3 days at 4C (to slow the clearing process)
Replace clearing solution with 80:20 of 1% KOH to glycerol and clear overnight at room temperature.
Replace clearing solution with 50:50 of 1% KOH to glycerol and clear overnight at room temperature.
Replace clearing solution with 20:80 of 1% KOH to glycerol and clear overnight at room temperature.
The sample can be stored indefinitely in 20:80 of 1% KOH to glycerol.