university of california, san diego histology shared resources

Decalcification Protocol
The following protocol is to be used for bone sample processed for paraffin embedded light microscopy
with H&E staining.
Equipment and Materials:
Styrofoam board or cork cutting board
Scissors, delicate ½”—Fisher Cat. No. 08-951-5,
Or Razor Blades, or scalpel
Forceps, curved—VWR Cat. No. 25714-007
Secureline Marker—Fisher Cat. No. 14-905-30 (pencil is also acceptable)
HistoPrep Tissue Capsules—Fisher Cat. No. 15-182-219
Cal-Ex –Fisher Cat. No. CS510-1D in glass jar or specimen cup
Or Cal-Ex II –Fisher Cat. No. CS511-1D in glass jar or specimen cup
Personal protective equipment e.g. lab coat, gloves, eye protection.
Bone decalcification
Generally the hind limb is taken during autopsy and fixed in formalin for a minimum of two days. At the
time the organs are being trimmed for processing, it will be useful to prepare the bone for decalcification
at this step.
1. With a scalpel or other cutting instrument, remove all of the skin and muscles taking care not to
remove the cartilage around the knee joint or scraping the bone too hard. (The skeletal muscles must
be removed as the decalcifying solution does not penetrate through the muscle.)
2. Place the bone in the appropriate cassette (see Blocking Chart) and immerse in container of Cal-Ex at
room temperature. No agitation is required but can be useful.
3. If the limb is not prefixed, it may be placed directly into Cal-Ex II. This solution is a combination of
fixative and decalcifyer and takes no more that two days for both processes to take place. As above,
the skeletal muscles and skin must be removed for the solution to take effect and placed in the
appropriate cassette.
4. For mouse bones, decalcification should not go beyond 2 days as overdecalcification will occur, tissue
basophilia will be lost, and poor hematoxylin staining will result.
5. To halt the decalcification process and to prepare specimen for processing, wash in running tap water
for approximately 2 hours.
6. The tissue is now ready to be placed in 70% alcohol and ready for processing.
As with the other mouse organs, it recommended that the bones do not remain in alcohol longer that
overnight.