Immunohistochemical and Immunocytochemical Localization of Amylase in Rat Parotid Glands and von Ebner's Glands by Ion Etching-Immunoscanning Electron Microscopy

Abstract

The distribution of amylase in rat parotid glands and von Ebner's glands was examined using ion etching-immunoscanning electron microscopy, which enables both light and electron microscopic observations of identical semi-thin resin sections immunolabeled with anti-α-amylase and immunogold in association with silver enhancement. At the light microscopic level, most acinar secretory granules (SG) and striated duct secretions of parotid glands were strongly stained dark brown. In von Ebner's glands, acinar SG and duct secretions were weakly to strongly stained light to dark brown. At the electron microscopic level, labeling was observed as bright gold-silver particles. The labeling intensity of acinar SG of parotid glands was higher than that of von Ebner's glands. In parotid glands, weak labeling of SG in transitional cells between acini and intercalated ducts, very weak labeling of SG in intercalated ducts, and strong labeling of striated duct secretions were observed. In von Ebner's glands, the secretions and some SG of interlobular ducts were strongly labeled compared to those of intralobular ducts and SG of acini. Less amylase was synthesized in von Ebner's acini compared to parotid acini, whereas von Ebner's ducts may secrete significantly more amylase to modify saliva than parotid ducts.

Keywords: amylase; localization; parotid gland; scanning electron microscopy; von Ebner’s gland.

Fig. 1

(A & B) Immunolight micrographs of rat parotid gland. Secretory granules in acinar cells in Figure 1A and secretory materials in striated duct cavity in Figure 1B are strongly stained dark brown for amylase by protein gold labeling with the silver enhancement method; 2,000×. (C) Immunoscanning electron micrograph of rat parotid gland. Ion etching was performed on the immunostained section. Same area as Figure 1A. Acinar SG are labeled with bright gold-silver particles for amylase. Unstained portion of Figure 1A is recognizable as intercalated duct (ID) that has dark small SG in apical cytoplasm. N: Nucleus; 4,000×. Bars=10 µm.

Fig. 2

IE-immunoSEM of rat parotid gland. High magnification of the top boxed region in Figure 1C. Parotid acinar typical SG (PA1 SG: 0.5–1 µm in diameter) are strongly labeled with many electron-reflective gold-silver particles (100 nm in diameter). Dispersion of labeled contents is seen at lumen (arrowheads). Labeled spherules are discharged at the apical membrane of the cell (arrows). Labeling particles over Golgi apparatus (GA) are few; 15,000×. Bar=1 µm.

Fig. 3

IE-immunoSEM of rat parotid gland. High magnification of the (A) middle and (B) bottom boxed regions in Figure 1C; 15,000×. (A) Strong labeling of peripheral zone with somewhat central zone in parotid acinar occasional SG (PA2 SG) is observed. A few particles over some small SG (arrowheads) are observed in the apical cytoplasm of intercalated duct (ID). At the luminal membrane, some labeled SG are exocytosed by merocrine secretion (double arrowheads). GA: Golgi apparatus, N: Nucleus. (B) Weak labeling of SG of a transitional cell (TC) adjacent to the ID cell. The SG in TC are larger and more electron-reflective than the SG in the ID cell. Bars=1 µm.

Fig. 4

IE-immunoSEM of rat parotid glands. (A) Intercalated duct (ID). Secretory material on apical membranes is labeled sporadically. Arrowhead indicates a few labeling particles on apical small SG; 12,000×. (B) Striated duct cavity. Same as the boxed region in Figure 1B. Secretory material in luminal cavity is labeled strongly, but spherules on luminal membrane are unlabeled. N: Nucleus; 15,000×. (C) Control involving anti-amylase absorbed to amylase displays few labeling. In usual acinar cells, SG are filled with amorphous structures and the contents are dispersed into the luminal cavity (arrowheads). In occasional acinar cells, some SG contain peripheral ring structures (double arrows). Spherical material is excreted (arrow). GA: Golgi apparatus; 12,000×. Bars=1 µm.

Fig. 5

Labeling density of amylase in the cellular compartments of (A) parotid glands and (B) von Ebner’s glands. Each of typical acinar SG was considered as control and compared to other cellular compartments. (**P<0.01, ***P<0.001). D SG: duct secretory granules, D SM: duct secretory material, EA1: von Ebner’s acinar typical, EA2: von Ebner’s acinar occasional, PA1: parotid acinar typical, PA2: parotid acinar occasional.

Fig. 6

ImmunoLM of rat von Ebner’s glands; 2,000×. (A) SG in most acinar cells shown on the left and secretory material in intralobular duct cavity shown on the right side of the broken line are weakly stained light brown. SG in some acinar cells are moderately stained brown. (B) The contents of the interlobular duct cavity are strongly stained dark brown. Bars=10 µm.

Fig. 7

IE-immunoSEM. Identical areas with the (A) left and (B) right half sides in Figure 6A. N: Nucleus; 4,000×. (A) Acini. Moderate labeling of von Ebner’s acinar occasional SG (EA2 SG) in the boxed region on the right side. Weak labeling of other von Ebner’s acinar typical SG (EA1 SG). (B) Intralobular duct. Apical dark vesicles and weakly labeled SM are observed. Bars=10 µm.

Fig. 8

IE-immunoSEM. Identical area with Figure 6B. Interlobular duct. Apical dark vesicles and strongly labeled SM are observed. N: Nucleus; 4,000×. Bar=10 µm.

Fig. 9

IE-immunoSEM of rat von Ebner’s glands; 12,000×. Identical areas with (A) left and (B) right boxed regions in Figure 7A, (C) the boxed region in Figure 7B, and (D) the boxed region in Figure 8. (A) Typical acinar cell. Weak labeling within EA1 SG. Labeling particles over Golgi apparatus (GA) are few. (B) Typical acinar and occasional cells. In both cases of weak labeling on EA1 SG and moderate labeling on EA2 SG, particles are concentrated within each acinar SG. (C) Apical cytoplasm of an intralobular duct. Weak labeling on SM of duct cavity and very weak labeling on a small SG (arrow). (D) Apical cytoplasm of an interlobular duct cell. Strong labeling on SM and some small SG (arrows). Weak labeling on other small SG (arrowheads). No labeling on vesicles (v). Bars=1 µm.