In my experience decalcification of rat bones (tibiae and femurs) in 14% EDTA (pH 7.2) takes much, much longer than 7-10 days (this is how long it would take for mouse bones, but rat bones are obviously much bigger). Using EDTA it usually takes several weeks to fully decalcify rat bones at 4 ºC.

Thanks, Thomas, 

Thanks for sharing your protocol,

Best,

Subhash

There is another method using acid decalcification with endpoint analysis to ensure complete decalcification. Let me know if you are interested in this methods. The advantage is speed and assurance that decalcification is complete. The disadvantage is that it may affect antigens which may affect immunohistology (but not all antigen-antibody binding is affected and the other stains you are interested work well).

At first collect your bone sample and keep it in 4% PFA for 24hr. Then transfer the bone sample into 70% Isopropanol for another 24hr. Now keep the bone into 8% EDTA solution, place it in 37* C incubator. change the EDTA solution every 5-6 days until the bone become soft. Store the demineralize bone at 4*C in 70% Isopropanol before use. Follow normal paraffin sectioning protocol to prepare thin section.

Decalcification Solutions for Bone

Calfor and XL-Cal Immuno Bone Decalcifier

Choose of decalcification solution will depend on the type of sections to be cut, type of staining to be performed, speed of process required, and it may be necessary to experiment with different solutions to obtain the best results with your material and staining method. The following decalcification solutions have been found useful for various purposes.

5%-10% Nitric Acid in Distilled Water

This solution gives the quickest results, but the large bubbles that form during decalcification can severely disrupt cells. This solution can be used when you need to make sections quickly. Specimens up to 0.5 mm thickness can be decalcified in 0.5-2 hours, while very thick specimens may require up to 6 hours or more at room temperature for complete decalcification. Decalcify until no bubbles are visible when the material is examined under a dissection microscope, but check thicker specimens thoroughly as a still calcified core may remain after all bubbles cease. After declacification is complete, the tissues should be neutralized by sodium sulfate for 12 hours before dehydration by an ethanol-xylene series.

10% HCl in Distilled Water

This solution can be used when you need to make sections quickly. Decalcification time varies depending on size of specimens and it may require 2 to 6 hours or more at room temperature for complete decalcification. The tissues decalcified with this solution should be neutralized by sodium sulfate for 12 hours before dehydration by an ethanol-xylene series.

Plank-Rychlo’s Solution

0.3 M Aluminium Chloride, 3% HCl, and 5% Formic Acid; Plank and Rychlo 1952

5% Trichloracetic Acid (TCA) in Distilled Water

Some studies suggest good TUNEL staining of the maxilla treated with 5% TCA. Based on the required time for processing and the signal-noise ratio, it is recommended 5% TCA as the decalcifying reagent to analyse for DNA strand breaks.

Morse’s Solution (10% Sodium Citrate, 20% Formic Acid)

Morse's solution is recommended to decalcify tissues to be processed for the rapid ISH analysis of specific RNA expression. A study shown that it detected specific mRNAs strongly in sections treated with Morse's solution.

5% Formic Acid (FA) in Distilled Water

Acid decalcification such as formic acid is generally quicker than EDTA chelation but studies have suggested that it may result in hydrolysis of DNA. However, some studies have shown that limited acid decalcification (less than 24 hr) in 5% formic acid can preserve DNA sufficient forfluorescent in situ hybridization (FISH) or comparative genomic hybridization (CGH) and that prolonged 10% formic acid decalcification results in failure of FISH and only limited retrieval of DNA for CGH studies. This method is also suitable for some immunohistochemical staining protocols.

10% EDTA (pH7.4), or 10% EDTA/TRIS-HCl (pH 7.4), or 10% EDTA with 0.07% (w/v) Glycerol (pH 7.4)

EDTA is a chelating agent, and it can be made 10% solution with distilled water, pH 7.4. This is also the preferred solution for decalcifying bone material for transmission electron microscopy. Specimens can be decalcified in this solution over several days up to several weeks in a refrigerator at 4 C, depending on degree of mineralization and size of specimen. The fresh solution is changed several times or once a week. After decalcification, samples can be routinely processed and embedded in paraffin.

Several studies have shown that EDTA decalcified bone material preserves DNA better, and preferable for ISH analysis, and TUNEL staining. It is also suitable for most of immunohistochemical staining protocols.

hello guys

there is another protocol for bone decalcification which is faster then other as below:

Protocol for bone decalcification:-

Material:

Prepare 10% paraformaldehyde (PFA), 6% Trichloroacetic acid.

Method:

1-    Collect the required tissue in appropriate manner.

2-    Immediately place tissue in chilled 10% PFA for 24 hr – preferably do this in a fume hood. The volume of PFA should be approximately 10X the volume tissue.

3-    Place the tissue in 6% trichloroacetic acid for 2 days.

The calcium salt will dissolve and the bone will be flixable

Dear collegues!

In all protocols, the bone at first fixed in 4% formaldehyde. But nowhere is it written that is it fixation in buffer solution (phosphate, cacodylate) or in distilled water.

The second question - if I want to use samples for electron microscopy can I directly fix them in a mixture of formaldehyde and glutaraldehyde (before EDTA treatment) or this prefixation before EDTA treatment (177 g/l in water) with formaldehyde 4% alone is better?

Rustem Uzbekov

bone should be fixed in 4% formaldehyde in phosphate buffer. If you want to use sample for electron microscopy mixture of formaldehyde and glutaraldehyde is better fixative before EDTA treatment. 

In my experience decalcification of rat bones (tibiae and femurs) in 14% EDTA (pH 7.2) takes much, much longer than 7-10 days (this is how long it would take for mouse bones, but rat bones are obviously much bigger). Using EDTA it usually takes several weeks to fully decalcify rat bones at 4 ºC.

It will definitely takes more time (than 7 days) if the tissue is with EDTA in 4 degree C.

As per this paper, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3040998/pdf/TODENTJ-4-223.pdf,

told 42 degree, enhance the delcacification with better preserved protein and nucleic acid stability. 

Hi Subhash,

I think tha this paper could be help to you, too. You look at it.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3040998/pdf/TODENTJ-4-223.pdf

Other method is old - Morse method and for quickly decalcification Shipley method.

Best regards,

Milos 

P.S. Excuse my English. thanks.