Substructures of the acinar basement membrane of rat submandibular gland as shown by alcian blue staining and cryo-fixation followed by freeze-substitution.

The ultrastructure of the epithelial basement membrane was studied in the acinar cells of adult rat submandibular glands after: (i) immersion fixation in 0.1 M sodium cacodylate buffer (CB, pH 7.2) containing 2.5% glutaraldehyde (GA) and alcian blue (0.5%) in conjunction with microwave irradiation, (ii) perfusion fixation with 2.5% GA in CB, (iii) rapid freezing followed by freeze-substitution (RF-FS) with 1% GA in acetone, and (iv) RF-FS with 2% OsO4 in acetone. The specimens were post-fixed with 1% OsO4 in CB after methods (i) and (ii) but not (iii) and (iv). Fixed specimens were embedded in epoxy resin and the ultrathin sections were cut, stained with both lead and uranium, and observed under a transmission electron microscope. Various substructural components could be seen in the acinar basement membrane. In the specimens processed by method (iv), a clear meshwork structure could be found just beneath the basal plasma membrane. This meshwork could not be seen in the specimens processed by method (iii) but thin filaments of approximately 100 nm in length extending from the epithelial base toward the connective tissue space were evident. By methods (i) and (ii), an electron dense 30-75 nm layer could be seen subjacent to basal cell membranes. By method (ii), particularly, thick threads connecting this layer to collagen fibres in the connective tissue were stained with alcian blue. Lamina lucida was not identified.