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

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.