Animal Tissue Techniques

Fourth edition 
Gretchen L. Humason 

Decalcification 

Calcium deposits may be so heavily concentrated in the tissue that they may interfere with sectioning and result in torn sections and nicks on the knife edge.  If deposits are sparse, overnight soakings of blocked tissue in water will soften the deposits sufficiently for sectioning.  Heavy deposits may be removed by any of several methods, but do not leave tissue in any fluid longer than necessary. 

If any doubt arises about the completion of decalcification, check for calcium by the following method: 

To 5ml of the solution containing the tissue add 1ml of 5% sodium or ammonium oxalate.  Allow to stand for 5 minutes.  If precipitate forms, decalcification is not complete.  A clear solution indicates it is complete.  Sticking needles in the tissue to check hardness is a sloppy technique that can damage cells. 

An excellent decalcifying fluid, RDO can be purchased by the gallon.  After using RDO for several years,  I recommend it as superior to other solutions.  Its rapidity of action is remarkable and the quality of staining and histological detail following its use is excellent.  Old bones cut down to one centimeter in thickness, if possible, require a six hour treatment: small and young pieces, only one to two hours.  Teeth will require overnight and up to eighteen to twenty four hours.  Do not over decalcify; this detracts from the staining quality.  Decalcifying may be followed by a brief washing in water but this is not necessary.  Fixation and decalcification may be combined in a mixture of one part undiluted formalin with nine parts RDO. 

 
Manufacturers Note: 

The combination of RDO and formalin is discourage, but should always be done under a fume hood to ensure the removal of potentially harmful vapors  Always follow the suggested directions for use.