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Int J Clin Exp Pathol 2013;6(10):1972-1983

Original Article
Assessment of different decalcifying protocols on Osteopontin and Osteocalcin
immunostaining in whole bone specimens of arthritis rat model by confocal

Susana A González-Chávez, César Pacheco-Tena, Cristina E Macías-Vázquez, Eduardo Luévano-Flores

Facultad de Medicina, Universidad Autónoma de Chihuahua, Chihuahua, Chihuahua, Mexico; Química Agronómica de México, Parque
industrial Impulso, Chihuahua, Chihuahua, Mexico; Departament of Pathology, Hospital CIMA Chihuahua, Chihuahua, Chihuahua, Mexico

Received August 9, 2013; Accepted September 3, 2013; Epub September 15, 2013; Published October 1, 2013

Abstract: Confocal immunofluorescence is a valuable technique for the detection of relevant molecules in the pathogenesis of arthritis in rat
models; however, it requires efficient processing of tissues including bone decalcification. The decalcification process must ensure the
complete removal of calcium and also a proper preservation of cellular structures and, specially, the antigenicity of the tissue to allow the
immunodetection of the molecules of interest. In the present study, we evaluated the effect of four different decalcifying solutions: the Morse´s
solution, 10% EDTA (pH 7.4), 7% HCl/2% EDTA and 5% Nitric acid, as well as four different treatments of the tissues (including microwave
irradiation) in the processes of decalcification for large pieces of adult rat bones (hind paw, fore paw, knee and column). We assessed the time
of decalcification, the easiness of slicing, the morphological preservation and finally, the antigenicity of two different bone proteins (Osteopontin
(OPN) and Osteocalcin (OC)) measured by its immunofluorescence intensity under controlled confocal microscopy conditions. Our results
showed that the specimen size and the presence of skin are critical factors for the rate of decalcification, and no significant benefit was found if
microwave irradiation is applied to the tissue. The comprehensive statistical analysis showed that the optimal solution for the detection of OPN
and OC by confocal immunofluorescence is the 5% Nitric Acid, and followed by 10% EDTA (pH 7.4), Ana Morse solution and 7% HCl/2% EDTA.

Keywords: Bone decalcification, arthritis rat model, confocal immunostainig

Address correspondence to: Susana A González-Chávez, M.Sc., PABIOM Laboratory, Facultad de Medicina, Universidad Autónoma de
Chihua-hua. Circuito Universitario Campus II, Chihuahua, Chihuahua, Mexico, CP 31125. Tel: (52) 614-2386030 ext. 3586; E-mail: