We examined the immunocytochemical localization of amylase in cryofixed serous acinar cells of gerbil major salivary glands by indirect immunostaining, using anti-gerbil parotid amylase antibody and protein A-gold complex. Fresh tissue blocks were quickly frozen by the metal-contact method, using liquid helium, and were freeze-substituted with either osmium-acetone solution or glutaraldehyde-containing acetone. They were then embedded in an epoxy resin mixture which was polymerized at 60 degrees C. Some tissue blocks substituted with aldehyde-acetone solution were embedded in Lowicryl K4M, polymerized at -30 degrees C. Thin sections of epoxy resin-embedded materials were treated with an oxidizing agent before immunostaining. The labeling density on the materials processed by various protocols for preparatory procedures was quantitatively compared to examine the usefulness of application of cryofixation to immunocytochemistry. The central dense core of heterogeneous secretory granules in the serous acinar cells of the parotid and sublingual glands was heavily labeled with immunogold, regardless of substitution media and embedding resins employed. The immunolabeling pattern clearly distinguished between the dense core and the surrounding matrix. Labeling density in the cryofixed materials was about 1.5 times greater than in those processed by conventional chemical fixation. Seromucous secretory granules in the submandibular gland acinar cells were only faintly labeled. The results obtained indicate that application of immunostaining to quick-frozen, substitution-fixed tissues is useful for high-resolution immunocytochemistry.
Two cytosolic carbonic anhydrase isozymes (CA-II and CA-III) were studied by immunohistochemistry in bovine parotid glands during fetal development. In a 3-month-old fetus of crown-rump length (CRL) 17 cm, the expression of CA-II in undifferentiated epithelial cells was observed, whereas immunostaining for CA-III remained negative. At 26 cm CRL (4-5 months old), weak expression of CA-III in large ductal epithelial cells was noted. The accumulation of secreted granules in primary acinar cells was initially observed at this stage. In a newborn calf, anti-CA-II reactivity almost disappeared from most duct segments. The time-dependent expression and distribution of the isozymes in parotid glands may reflect different biological functions of these structurally closely related isozymes. Bovine parotid acinar cells of fetuses would thus appear to possess all the cellular structures and immunohistochemical properties at 4 and 5 months of gestation. CA-II subsequently disappeared from duct segments and nearly all acinar cells in adults were present at or just after birth.
The immunolocalization of carbonic anhydrase (CA) isozymes in bovine salivary glands and stomach were first investigated in order to discuss their biologic functions. In parotid glands, CA-II was located in serous acinar cells, whereas CA-III, in the duct segments. In contrast, a strong reaction was shown for both CA-II and CA-III in the duct segments of the submandibular gland, especially CA-III was selectively located in basal cells of the interlobular ductal epithelium; however, these glands essentially lacked CA-I. On the other hand, epithelial cells of the rumen, reticulum and omasum showed cytoplasmic reaction for CA-I, II and III in all layers of the epithelium, except the stratum coroneum. The parietal cells in the abomasal epithelium were more intensely stained for CA-II, but not for CA-I and CA-III. Immunolocalization of CA isozymes in serous cells in the parotid gland indicates their primary function in secreting macromolecules, whereas localization of CA in striated and excretory ducts in the parotid and submandibular gland suggests their traditional function in fluid and electrolytic transport. The biologic function of CA isozymes in the ruminal, reticular and omasal mucosa are postulated to influence the absorption and excretion of volatile fatty acid and NH4; the abomasal parietal cell is considered to be involved in ion transport.
Immunohistochemical localizations of carbonic anhydrase isozymes (CA-I, CA-II and CA-III) in equine and bovine digestive tracts were studied. In the horse, epithelial cells in both the oesophagus and non-glandular part of the stomach lacked all three isozymes. In contrast, surface epithelial and parietal cells in the glandular region of the stomach showed reactivity for CA-II. In the small intestine, absorptive columnar cells covering the villi in the duodenum were positive for CA-II. The epithelium of the jejunum and ileum lacked all three isozymes. In the large intestine, CA-II was detected in the columnar cells in the upper part of the crypt. In cattle, epithelial cells of the oesophagus showed reactions for CA-I and CA-III but not for CA-II. Although the absorptive epithelial cells of the small intestine lacked CA-I, CA-II and CA-III, those of the upper part of large intestine crypts were heavily stained for all three isozymes.
The immunolocalization of carbonic anhydrase isozymes in equine salivary glands was investigated for assessment of their biologic functions. In parotid glands, duct segments showed reactivity with CA-I and CA-III. CA-III was selectively located in duct segments, particularly in the basal cells of the interlobular duct. Serous acinar cells were positive for CA-I and CA-II. In submandibular glands, CA-I and CA-II were present in serous demilune and duct segments. CA-II was selectively located in the duct segments, as also noted in the parotid gland. In sublingual glands, CA-I and CA-II were located in serous demilune, as also in the case of the submandibular gland. In the duct segments, all the isozymes considered in this study were found to be present.
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