In vivo

Cleared Skeletal Preparations 

In vitro

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

Solutions:

  • -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

Procedure:

  1. At autopsy, place embryo in tap water for 1-5 hours (optional)
  2. Scald embryo in hot tap water (~65C) for 20-30 seconds for easier maceration of the tissue
  3. Use forceps to eviscerate the embryo.
  4. Fix embryo in 95% EtOH overnight. Be sure to remove all bubbles from the body cavity
  5. Transfer embryos to 100% Acetone and incubate overnight at room temperature
  6. 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
  7. Rinse embryos twice in 95% EtOH. Destain embryos in 95% EtOH overnight at room temperature.
  8. Slightly clear the sample by placing in 1% KOH for 1 hour.
  9. Counterstain bone in Alizarin Red stain for 3-4 hours.
  10. 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.