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A Modified Protocol for Staining of Undecalcified Bone Samples Using Toluidine Blue—A Histological Study in Rabbit Models

et al. Stefan Peev

Applied Science


Undecalcified bone histology is a valuable diagnostic method for studying bone microarchitecture and provides information on bone formation, resorption, and turnover. It has various clinical and research applications.

Toluidine blue has been widely adopted as a staining technique for hard-tissue specimens. It provides a clear identification of bone structural and cellular features and the distinctions between them.

Furthermore, the method allows for an excellent definition of the cement lines that mark the fields of bone remodeling.

Some of the suggested and currently used processing and staining protocols are too complex and time-consuming, which necessitates their modification and/or optimization.

This research aims to develop a simplified protocol for staining plastic-embedded undecalcified bone specimens with toluidine blue.

The samples were obtained from the tibial bones of rabbits, and experiments with and without pre-etching were conducted.

Our results demonstrated that the optimal visualization of the bone microstructure and its cellular components was achieved in the samples without acid pre-etching and dehydration after staining.


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