The comparative analyses of decalcification procedures and methyl benzoate pre-treatment on tissue preservation and antigenicity in human acetabular labra

Abstract

The histological processing of musculoskeletal tissue might be challenging. The alteration of tissue composition e.g. by calcification of soft tissue in the elderly, after trauma or surgical interventions makes the histological processing of fixed tissue difficult. Additional steps of decalcification are then needed that probably affect the staining quality. In the present work, the effects of different decalcification agents and the intermedium methyl benzoate on histological staining methods and immunohistochemistry have been compared. Acetabular labra were fixed with 4% paraformaldehyde, left untreated or decalcified using 30% ethylenediaminetetraacetic acid (EDTA; Chelaplex®) or 6% trichloroacetic acid (TCA) for 1-4 days to investigate the effects of decalcification duration. Moreover, samples were pretreated with methyl benzoate or conventionally paraffin embedded independent of decalcification procedure and duration. The specimens were evaluated using hemalaun-eosin, Azur II- methylene blue staining or immunohistochemistry against ankyrin B to visualize nerve fibers. Decalcification with Chelaplex® or TCA reduced cutting artifacts without affecting the tissue morphology and proteoglycan staining but decreased antigenicity in immunohistochemistry. Interestingly, methyl benzoate further reduced cutting artifacts without altering tissue morphology and elevated antigenicity for Chelaplex® decalcified tissue samples in immunohistochemistry. The decalcification with Chelaplex® or 6% TCA preserves tissue morphology and proteoglycan staining similar to non- decalcified tissue but facilitates section processing. In immunohistochemistry both decalcification agents decreased antigenicity. Chelaplex® decalcified, methyl benzoate treated samples yielded an improved antigenicity.