Comparison of ethylenediaminetetraacetic acid and rapid decalcificier solution for studying human temporal bones by immunofluorescence.

OBJECTIVES

The pervasiveness of hearing loss and the development of new potential therapeutic approaches have led to increased animal studies of the inner ear. However, translational relevance of such studies depends upon verification of protein localization data in human samples. Cadavers used for anatomical education provide a potential research resource, but are limiting due to difficulties in accessing sensory tissues from the dense temporal bones. This study seeks to reduce the often months-long process of decalcification and improve immunofluorescent staining of human cadaveric temporal bones for research use.

METHODS

Temporal bones were decalcified in either (a) hydrochloric acid-containing RDO solution for 2 days followed by 0.5 M ethylenediaminetetraacetic acid (EDTA) for 3 to 5 additional days, or (b) 0.5 M EDTA alone for 2 to 4 weeks. Image-iT FX signal enhancer (ISE) was used to improve immunofluorescent signal-to-noise ratios.

RESULTS

The data indicate that both methods speed decalcification and allow for immunolabeling of the extranuclear proteins neurofilament (heavy chain), myosin VIIa, oncomodulin and prestin. However, RDO decalcification was more likely to alter structural morphology of sensory tissues and hindered effective labeling of the nuclear proteins SRY-box transcription factor 2 and GATA binding protein 3.

CONCLUSIONS

Although both approaches allow for rapid decalcification, EDTA appears superior to RDO for preserving cytoarchitecture and immunogenicity.

LEVEL OF EVIDENCE: NA.