Visualization of the real microarchitecture of glycoprotein matrices with scanning electron microscopy

Preparation of biological samples for scanning electron microscopy considers fixation, dehydration and drying, such procedures strongly stress the delicate filamentous structure of glycoprotein matrices, due to their extremely hydration state. Therefore, in order to allow the observation of the real microarchitecture of these matrices, a specific technique was developed by us. Mucus covering the jejunum surface and the zona pellucida surrounding the mature oocyte were examined. The technique consists in the use of ruthenium red (RR), saponin, osmium and thiocarbohydrazide. RR prevents the dissolution and/or the alteration of glycoproteins and polyanionic carbohydrates induced by aqueous fixatives, whereas saponin is a detergent of soluble proteins. Osmium-thiocarbohydrazide preserves matrix filaments from the mechanical stress induced by dehydration and critical point drying, thus minimizing filaments packing and shrinkage.