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 for fluorescent 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.






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2. Verdenius HHW and Alma L (1958) A quantitative study of decalcification methods in histology J Clin Pathol. 1958 May; 11(3): 229–236.


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4. Janneke C. Alers, Pieter-Jaap Krijtenburg, Kees J. Vissers, and Herman van Dekken (1999) Effect of Bone Decalcification Procedures on DNA In Situ Hybridization and Comparative Genomic Hybridization: EDTA Is Highly Preferable to a Routinely Used Acid Decalcifier. Journal of Histochemistry and Cytochemistry, Vol. 47, 703-710


5. P Sarsfield, C L Wickham, M V Joyner, S Ellard, D B Jones, and B S Wilkins (2000) Formic acid decalcification of bone marrow trephines degrades DNA: alternative use of EDTA allows the amplification and sequencing of relatively long PCR products. Mol Pathol; 53(6): 336


6. Yasuaki Shibata, et al (2000) Assessment of decalcifying protocols for detection of specific RNA by non-radioactive in situ hybridization in calcified tissues. Histochem Cell Biol 113:153–159


7. Yamamoto-Fukud T (2000) Effects of various decalcification protocols on detection of DNA strand breaks by terminal dUTP nick end labelling. Histochem J. 2000 Nov;32(11):697-702.


8. Brown RSD, Edwards J, Bartlett JW, Jones C, and Dogan A (2002) Routine Acid Decalcification of Bone Marrow Samples Can Preserve DNA for FISH and CGH Studies in Metastatic Prostate Cancer. Journal of Histochemistry and Cytochemistry, Vol. 50, 113-116.


9. Recent Advances In Microwave Decalcification Protocols by Herbert Skip Brown.