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BioMed Research International
Volume 2017, Article ID 9050754, 6 pages
Research Article

Evaluation of Decalcification Techniques for Rat Femurs Using HE and Immunohistochemical Staining

1Preclinical Medicine School, Beijing University of Chinese Medicine, Beijing 100029, China
2Chinese Material Medica School, Beijing University of Chinese Medicine, Beijing 100029, China
3Diabetes Research Center, Beijing University of Chinese Medicine, Beijing 100029, China
4The Research Institute of McGill University Health Center, Montreal, QC, Canada H4A 3J1
5Oral Biological Medicinal Science, University of British Columbia, Vancouver, BC, Canada V6T 1Z3

Correspondence should be addressed to Dongwei Zhang; moc.liamg@6001iewgnod and Sihua Gao; moc.361@6121auhisoag

Received 16 November 2016; Accepted 20 December 2016; Published 26 January 2017

Academic Editor: Alexander N. Orekhov

Copyright © 2017 Haixia Liu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Aim. In routine histopathology, decalcification is an essential step for mineralized tissues. The purpose of this study is to evaluate the effects of different decalcification solutions on the morphological and antigenicity preservation in Sprague Dawley (SD) rat femurs. Materials and Methods. Four different decalcification solutions were employed to remove the mineral substances from rat femurs, including 10% neutral buffered EDTA, 3% nitric acid, 5% nitric acid, and 8% hydrochloric acid/formic acid. Shaking and low temperature were used to process the samples. The stainings of hematoxylin-eosin (HE) and immunohistochemical (IHC) were employed to evaluate the bone morphology and antigenicity. Key Findings. Different decalcification solutions may affect the quality of morphology and the staining of paraffin-embedded sections in pathological examinations. Among four decalcifying solutions, 3% nitric acid is the best decalcifying agent for HE staining. 10% neutral buffered EDTA and 5% nitric acid are the preferred decalcifying agents for IHC staining. Significance. The current study investigated the effects of different decalcifying agents on the preservation of the bone structure and antigenicity, which will help to develop suitable protocols for the analyses of the bony tissue.