I found the suitable procedure for the extraction of cellular debris from the glutaraldehyde fixed tissue.

But I have a problem in the tissue homogenisation procedure the

HEPES-sucrose buffer (buffer 1): contains:

20 mmol/L HEPES, 225 mmol/L sucrose,

1 mmol/L EDTA,

pH 7.4

protease inhibitors (5 µg/ml pepstatin & 5 µg/ml aprotinin)

I do not wish to add EDTA as it will chelate the calcium that is bonded on the cellular debris. And my main aim to quantify the bonded calcium before and after use of anti-calcification treatment of the tissue.

Can you please suggest me any alternative other than EDTA which wont affect the Calcium and which is safe for homogenisation..

Can I carry out this procedure without using Tris- EDTA..