Assessment of decalcification solutions on cellular morphology and immunostaining of mouse bones

Abstract

Decalcification is an important step in histology laboratories to allow mineralized tissue samples to be trimmed and sectioned easily. Many decalcifying solutions have a rapid onset of action in softening tissues but alter protein structure and morphology, while others preserve protein integrity but are less efficient. The ideal decalcification protocol allows for rapid and cost-effective processing and precise evaluation of microscopy and antigen-based immunostaining such as immunohistochemistry. In our study, mouse tissues were decalcified with three commercially available solutions to identify the product that best meets those criteria. ImmunocalTM (StatLab), EprediaTM, and Rapid-CalTM (StatLab) decalcification agents were tested on formalin-fixed, paraffin-embedded CD-1 mouse femur, skull, and sternum samples. Multiple metrics including ammonium oxalate turbidity and radiography were used to assess stages of bone demineralization. Tissues were routinely processed, embedded in paraffin, and sectioned at 5 µm for H&E staining and CD3, CD31, and Iba1 immunostaining. Each tested sample represented a decalcification product and time (4, 6, 12, 24, 30, 48, 73 h). Samples were assessed by radiolucency on X-ray and gross bone pliability prior to histologic processing, followed by histopathologic scoring for completeness of demineralization, preservation of tissue architecture, and antigenicity of tissue. All three commercially available decalcifying solutions are sufficient for rapid decalcification with preservation of tissue integrity, cellular detail, and immunogenicity.