Robin this is the protocol we use to showing new bone old bone. Procedure for Eosin Y 0.6 % For Staining Bone Specimen Chemicals EosinY, Sigma E-4382, 100% Ethanol, Phloxine B (Sigma P-4030) Orange G(sodium salt-Sigma O-1625) Preparation: Stock 0.6% Eosin Add 6g of eosin to 900ml 100% Ethanol and100 ml of H20; stir to dissolve. Add 50 ml glacial acetic acid adjust PH to 4.6 and 5.0. The color of the solution will change from opaque green to clear red. Stock 1% Phloxine B Solution: Dissolve 1g of Phloxine B in 100 ml distilled water and filter to make a 1% solution. Stock 2% Orange G Solution: Dissolve 2 g of Orange G in 100ml distilled water and filter to make a 2% solution. Working Solution Eosin 0.6% Make a working solution by adding 6 ml of 1% Phloxine and 6 ml of 2% Orange G to 238 ml of 0.6% eosin. Old bone dark orange. New bone light orange. Stain 15 to 30 seconds depending on what intensity you prefer.
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