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Introduction: Mucopolysaccharides are of great interest in intestinal research. Histochemical methods are often employed to identify mucins. Since it is in the nature of
mucins that they are “hard to hold onto” once they come into contact with water, a frequently used medium in histochemistry, there are a number of challenges that may decrease diagnostic accuracy. As the outcome of methods published for microscopic detection of mucosubstances proved to be unsatisfactory in our hands, we aimed at establishing a reliable and reproducible protocol of our own.
Materials and Methods: Histological samples were obtained during necropsy of piglets from various feeding trials. Several sampling, fixation and staining procedures were evaluated for their use in semiautomatic quantification and quality assessment of different mucus fractions simultaneous on one tissue section.
Results: Cryostat sectioning, subsequent fixation steps with heat, ethanol and modified Bouin0s solution, followed by triple staining with high iron diamine, alcian blue and periodic acid-Schiff turned out to be the best method to identify sulfomucin, sialomucin and neutral mucin simultaneous on one tissue section. This methodology resulted in very good morphology of goblet cells with mucin containing vesicles intact within the cells. Morphology was comparable to ultrastructural electron microscopical observations. Semiautomatic quantification of different mucins was examined in samples of animals (4 groups of 6 animals) from a feeding trial. No statistical differences were found between the groups.
Conclusion: From a technical point of view, reliable mucus quantification in paraffin embedded specimens appears not to be practicable. Assessment of mucus quality involves strictly tested procedures and, according to our experience, the best approach is a combination of cryostat sectioning with three subsequent fixation steps. Afterwards, triple staining with high iron diamine, alcian blue and periodic acid-Schiff reaction can be employed to identify sulfomucin, sialomucin and neutral mucin simultaneous on one tissue section.