Evaluation of different decalcifying agents on histochemical and immunohistochemical staining properties of canine osseous tissue

Background: The purpose of tissue processing is to strengthen the tissue and place it in a suitable environment for slicing without causing damage. In routine histopathology, decalcification of calcified tissues is a principal step before tissue processing. Thus, the aim of this study is to evaluate the effect of several decalcifying agents on morphological and antigenic preservation in canine mandibular tissue. Methods: Four different decalcifying solutions, including 5% nitric acid (NA), 10% and 20% formic acid (FA), and 10% ethylene diamine tetraacetic acid (EDTA), were employed to remove mineral salts from specimens harvested from mandibular osseous tissues of four healthy Mongrel dogs (32 samples). The solutions were compared regarding the decalcification time, ease of tissue slicing, morphological organization after Alizarin red S staining, and osteocalcin antigenic preservation. Results: Considering the short decalcification time and ease of sectioning, 5% NA) yielded better results than 10% and 20% FA and 10% EDTA (pH 7.4). In terms of the preservation of morphology and antigenicity of the tissue samples, 10% EDTA was found to be the most optimal solution, followed by 10% and 20% FA and 5% NA. Conclusion: Our findings support EDTA as a highly preferred choice for the decalcification of canine osseous tissue when aiming for immunohistochemistry, despite its time-consuming nature. However, for general histological staining procedures, 20% FA and 5% NA are preferred.