Procedure 3.1 Accumate™ RDO (Sigma Aldrich, www.sigmaaldrich.com, 800-325-3010) Rapid Bone Decalcification Procedure

Recipes for Microwave Staining Kits

This procedure is intended for use as a "stat" alternative to room temperature decalcification of compact bone specimens (e.g. femur head). Due to variability in bone density from one specimen to the next, 2-3 repeats of the final irradiation step may be necessary. It is critical to perform flexibility checks at the stated intervals to prevent over-decalcification and loss of nuclear detail. For optimal effectiveness, the specimens should be secured in a microwavable cassette rack and placed in 300 ml of Accumate RDO Rapid Decalcification Solution in a 600 ml microwavable container. Cassettes should be positioned in the rack so that they are located in the mid-portion of the Accumate RDO Rapid Decalcification Solution during the procedure. Agitation of cassettes between irradiation steps is a critical factor in penetration and performance of the decal solution. Bone specimens used should be no greater than 5 mm in thickness.

Note: This procedure is not intended for use on Cortical Bone (e.g. bone marrows), since cortical bone may be rapidly decalcified in Accumate RDO Decalcification Solution in 30-45 minutes.

Note: Bone must be fixed prior to decalcification with 10% Neutral Buffered Formalin.

PROCEDURE

1. Trim fixed bone specimen to 2-5 mm in thicknes, and place in a plastic cassette. Use one cassette per specimen and secure cassette(s) in a microwavable rack.
2. Pre-heat 300 ml of Accumate RDO Rapid Decalcification Solution at 800 watts for 25 seconds, without the rack/cassettes containing the tissue.
3. Place rack/cassettes containing tissue into the preheated Accumate RDO Rapid Decalcification Solution and agitate rack up and down 20 times. (Use air bubble agitation if available).
4. Place container containing rack/cassettes in the microwave cavity and microwave at 200 watts for 15 minutes.
5. Take container out of cavity in a well-ventilated area or under a fume hood and agitate rack/cassettes up and down 20 times.
6. Place container containing rack/cassettes in microwave cavity and microwave at 300 watts for 15 minutes.
7. Take container out of cavity in a well-ventilated area or under a fume hood and agitate rack/cassettes up and down 20 times.
8. Place container containing rack/cassettes in microwave cavity and microwave at 400 watts for 10 minutes.
9. Take container out of cavity in a well-ventilated area or under a fume hood and agitate rack/cassettes up and down 20 times.
10. Place container containing rack/cassettes in microwave cavity and microwave at 200 watts for 10 minutes.
11. Take container out of cavity in a well-ventilated area or under a fume hood and agitate rack/cassettes up and down 20 times. Determine decalcification endpoint by checking bone using standard methods. If decalcification is not complete, repeat final irradiation step (#10) until desired decalcification endpoint is acheived.

Note: It is critical to check the bone specimen prior to each repetition of final irradiation to prevent over-decalcification and loss of nuclear detail.

Note: This procedure should only be used in a laboratory microwave with a ventilation system.