Following this decalcification procedure for histology will result in calcium and mineral deposit removal without harming the bone or specimen you are working on!

This procedure for decalcifying bones and other specimens is most effective when used with RDO Decalcifier. Removing calcium and other mineral deposits is 100% essential for embedding.

RDO is a remarkably effective multipurpose decalcifier that can be tailored to suit your specific lab routine. Careful monitoring should be used with RDO to avoid over decalcification. Most decalcification occurs in approximately 4-6 hours or less, depending on the thickness and density of specimens. Overnight decalcification should be avoided. Full Strength RDO is used for hard compact bone, ie. femur heads. RDO may be diluted with tap water to permit multipurpose use. Dilution with tap water is recommended for mildly calcified specimens such as bone marrow biopsies (core). In either case, the standard procedure should be to check the specimen every ½ -1 hour for mildly calcified specimens and every 1-2 hours for compact bone to determine the endpoint of decalcification.

DECALCIFICATION PROCEDURE FOR HISTOLOGY

1. Tissues should be thoroughly fixed before decalcification. Most standard fixatives can be used prior to RDO use. To insure adequate fixation and decalcification, specimens should be trimmed to less than 1 cm thickness. Formalin fixation and RDO decalcification should not be combined. Hydrochloric acid (active ingredient of RDO) and formaldehyde vapors have been reported to form a potent carcinogen, bischloromethyl ether. Prior fixation with formalin is permissible. Brief washing in water before RDO decalcification is advised.
2. Do not use metallic equipment/cassettes for decalcification. RDO can pit or oxidize some metals after long periods of exposure. Decalcified tissues may be placed in metallic equipment after washing.
3. DO NOT OVER DECALCIFY. RDO action is very rapid. DO NOT leave bone specimen in RDO for several days as is required with commercial decalcifiers. MOST SPECIMENS CAN BE DECALCIFIED IN FOUR HOURS OR LESS. Use adequate volume of RDO to tissue; 20:1 volume ratio of RDO is recommended. The key determinations for time required for decalcification are size and density of the specimen. Most mature bones of 1 cm size are decalcified for 4-6 hours; smaller cancellous bone in 2-4 hours. Bone biopsies are decalcified in 30-60 minutes. Teeth and entire femur heads may require overnight treatment. If RDO action is too rapid, dilute with distilled or de-ionized water. Good results have been obtained on bone marrow biopsies with a 3:1 dilution (RDO to water).
4. Determine the end point of decalcification using standard methods (e.g. X-ray, flexibility, chemical analysis).
5. Proceed with routine processing and embedding. Washing tissue prior to processing is optional.
6. Overexposure to RDO can result in poor hematoxylin staining. If this occurs, satisfactory results can be obtained by treating deparaffinized slides prior to hematoxylin with aqueous saturated lithium carbon-ate (1-2 minutes) or 10% aqueous sodium bicarbonate (6-8 hours). Poor histological detail/artifacts (swelling, fragmentation) can occur from excess decalcification. Hemosiderin is not removed by RDO.

NEED TECHNICAL OR SDS INFORMATION?

Please click here for our Technical Page or here for our SDS!