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Effects of decalcification with 10% nitric acid solution and EDTA on Safranin O staining and aggrecan
Eduarda Correa Freitas1, Fernanda Visioli2, Luiza Falcão Vanzella3
1Tissue Bank Unit, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil.
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.
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