Effects of decalcification with 10% nitric acid solution and EDTA on Safranin O staining and aggrecan immunohistochemistry.

BACKGROUND: Decalcifying mineralized samples for microscopic analysis presents significant challenges. AIM: To evaluate the effects of different decalcification solutions on Safranin O staining and immunohistochemistry for aggrecan. MATERIALS AND METHODS: The paws of AG/WT, BALB/c, C57, DBA1/J mice, and Wistar rats were removed, fixed, and randomly divided into three groups: 10% nitric acid, 12.5% EDTA at room temperature, and 12.5% EDTA at 35°C with shaking. Tissue sections were prepared using a microtome and stained with Safranin O and an anti-aggrecan antibody. The staining intensity was then assessed. RESULTS: No statistically significant difference was found between the EDTA at room temperature and EDTA at 35°C for Safranin O staining. However, EDTA-treated samples generally exhibited stronger staining. For aggrecan quantification, the nitric acid protocol resulted in heterogeneous labeling among the studied strains. Notably, this protocol showed a statistically significant decrease in staining intensity in AG/WT and DBA1/J strains compared to the EDTA protocol. CONCLUSION: The EDTA protocol produced more intense Safranin O staining compared to nitric acid across all strains. In terms of aggrecan detection, the AG/WT and DBA1/J strains exhibited reduced immunohistochemical reactivity when decalcified with nitric acid compared to EDTA.