Alcian Blue staining of cartilage for electron microscopy. Application of the critical electrolyte concentation principle

Abstract

The critical electrolyte concentration principle was applied to the Alcian Blue staining of rat epiphyseal cartilage proteoglycans for electron microscopy. The distribution and structure of material in glutaraldehyde-fixed cartilage stained at pH 5.8 without MgCl2 and in the presence of 0.05, 0.4, 0.5, 0.9 and 1.0 M MgCl2 was compared with that produced by simultaneous staining and fixation at neutral pH. Both methods resulted in staining of intracellular material within vacuoles as well as staining of non-collagenous matrix material. The structure and distribution of Alcian Blue-positive matrix material consisted of rounded or polygonal granules which accumulated around cells in the proliferative and hypertrophied zones. A similar pattern of distribution was observed in samples stained in the presence of 0.4 or 0.5 M MgCl2. In these cases, however, the stained material exhibited a ribbon-like configuration and granules were few in number. Increasing the MgCl2 concentration to 1.0 M resulted in a marked reduction of Alcian Blue stained material. No ribbon-like structures were observed, and matrix granules were reduced in both number and size. The decreased staining associated with increased electrolyte concentration lends support to the concept that epiphyseal cartilage matrix granules are composed primarily of chondroitin sulphate, and suggest that this same material is present in vacuoles associated with the Golgi apparatus in chondrocytes of the proliferative and hypertrophying zones.