Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
Decalcification: Acid or EDTA?
How should I decalcify a bony specimen or a tooth? What precautions are needed if galactosidase activity must be preserved (to identify cells carrying the LacZ gene)?
Decalcification with EDTA is probably the best method with your LacZ, due to the enzyme staining you are doing. I would be careful to adjust the pH of the EDTA solution to the working pH of enzyme staining in PBS or a TRIS buffer, and rinse carefully in buffers postdecalcification. Formic acid may ruin LacZ enzyme staining results.
If the bone is crunchy, you have either not removed all the bone mineral, or you have transferred the bones from EDTA to alcohol and have precipitated EDTates in your tissue.
When you decalcify, do you determine the end point using an x-ray/calcium oxalate/prod with a pointed stick?
How long do you decalcify? Even at 20% EDTA these would take at least a week with vigorous agitation at room temperature. Is the EDTA buffered to pH 7? If not, you are using the solution as an acid decalcifier as well as a chelator. In this case, assuming your stain still works and will not be affected by acid pH, change to 10% formic acid, which provides much faster decalcification. Check the endpoint (when all the calcium is gone) daily.
[But see Answer 1 for acid-sensitivity of galactosidase.]
If you have checked the endpoint and all the calcium is gone, rinse the tissue in water for at least 8 hours to remove all the excess EDTA before putting it in alcohol.
Answer 3. (A formic acid procedure for teeth, with oxalate
The protocol we use here at Ind. Univ. School of Dentistry is as follows:
After teeth are fixed in 10% neutral buffered formalin, they are placed in wide mouth bottles with a 5% formic Acid solution. They are then checked each day by pipetting 5 ml of the acid solution into a test tube to which 1 ml of 2.5% ammonium oxalate is added. If a white precipitate forms there is still calcium present. The solution is then changed and the process repeated the next day. Once I get one negative test the specimen is grossed as needed and placed back into acid until another negative is obtained. The specimen is then placed in running water overnight and processed with the next days run. I know this can take a long time, but the results are worth it. If you need anything else let me know.
Lee Ann Baldridge
IUSD Oral Path Group