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Resolution and Discovery
Authors: Nada Žnidaršič, Polona Mrak, Eva Rajh, Kristina Žagar Soderžnik, Miran Čeh, and Jasna Štrus

Biomineralized structures consist of an organic matrix and mineral constituents. Imaging of the mineralized biological tissues is demanding due to specific requirements for the preservation and visualization of chemically different constituents and due to sectioning difficulties. In this study, a characterization of the cuticular matrix of the crustacean exoskeleton was performed by a combination of microscopic methods, aiming to obtain spatial information on the matrix composition. Histochemical procedures were performed and compared in artificially decalcified and non-decalcified samples, in paraffin and resin sections. Wheat germ agglutinin (WGA) lectin-gold conjugate and a fluorescent chitin-binding probe were used to localize chitin in paraffin and resin sections of samples prepared by different fixations. Calcified regions of the matrix were determined by histochemical staining of aldehyde-fixed, methanol-fixed, and resin-embedded samples and by scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM–EDXS) of “intact” cuticle transverse profiles that were not subjected to any surface processing. We show that the spatial distribution of cuticular organic matrix components is not homogenous, as a differential staining of exocuticular and endocuticular matrix was obtained by histochemistry. Chitin localization, performed by two different methods, shows a localization pattern of horizontal lines of alternating intensity in the transversely cut endocuticle. Histochemical demonstration of calcified cuticular matrix in resin sections was successful in 2.5 μm thick resin sections of methanol-fixed samples. Methanol-fixed, dried, and fractured samples displayed the characteristic elemental composition and are useful to obtain qualitative composition data from the non-processed surface profiles of the cuticle.

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