Alcian blue staining of glycosaminoglycans in embryonic material: effect of different fixatives

Glycosaminoglycans are important components of the extracellular matrix of developing embryos where they are found in the form of proteoglycans. Alcian Blue staining of tissue sections is the technique most commonly used for demonstrating their distribution. Glycosaminoglycans have a high solubility in water, and are easily lost from the tissue during processing, even if non-aqueous fixatives have been used. Formalin and Carnoy's fluid are the most frequently used fixatives, and the addition of cetyl pyridinium chloride has been recommended to reduce glycan solubility. Using sections of day-10 rat embryos containing developing head and heart (both known to be rich in glycosaminoglycans) the effects of ten fixatives have been investigated with and without cetyl pyridinium chloride on the preservation of Alcian Blue-stainable material (at pH 2.5) and tissue structure. The most useful fixatives were Karnovsky's and Sainte-Marie's. Both gave a strong and reproducible staining pattern of the extracellular polyanionic material. Sainte-Marie's gave better preservation of tissue structure, allowing the demonstration of cell-matrix interrelationships; Karnovsky's gave a better contrast between extracellular and intracellular staining, which is particularly useful at lower magnifications. Cetyl pyridinium chloride is a detergent. Transmission electron microscope observations showed that it causes cell membrane disruption and vesicle formation, which at the light microscopic level, would cause cell membrane-associated glycosaminoglycans to appear as stained strands wholly within the extracellular domain. Therefore the use of cetyl pyridinium chloride is inadvisable where a distinction between surface-related and extracellular glycosaminoglycans is desirable. It has the further disadvantage of enhancing cytoplasmic and nuclear polyanionic material, thus decreasing the differential staining intensity of intracellular and extracellular domains.