Evaluation of EDTA and nitric acid solutions for decalcification of joints in AG/WT, BALB/c, C57, DBA1/J mice, and in Wistar rats

Decalcification of mineralized samples for microscopic analysis involves competing factors including decalcification time, preservation of tissue integrity and cost. We investigated the utility of different decalcification solutions for studying joints in AG/WT, BALB/c, C57, DBA1/J mice and Wistar rats. The hind paws of the rodents were removed and fixed with 10% buffered formalin. Specimens were divided randomly into three groups for demineralization: 10% nitric acid, 12.5% EDTA at room temperature and 12.5% EDTA at 35 °C with shaking. Sections of joints were stained with hematoxylin and eosin (H & E). We evaluated decalcification time and expense, ease of cutting sections, preservation of nuclear basophilia and intranuclear detail, and intensity of eosin staining. The 10% nitric acid solution produced the most rapid decalcification for the mice, but not the rats. The 12.5% EDTA solution at 35 °C with shaking did not decrease decalcification time. Effects on microtomy were variable as were the effects on H & E staining. The EDTA solution provided the best basophilia and intranuclear detail for the mice. For rats, only 12.5% EDTA at 35 °C with shaking produced good preservation. Preservation of nuclear basophilia and intranuclear detail for rats was best with 10% nitric acid and EDTA 35 °C. For mice, 10% nitric acid failed to preserve nuclear basophilia and intranuclear detail. For intensity of eosin staining, EDTA at room temperature and EDTA 35 °C was best for both mice and rats. Sections also exhibited good H & E staining in most samples decalcified with 10% nitric acid. Although we found considerable variation among groups of animals, we found less variation among the different mouse strains than between mice and Wistar rats.