J Biomedical Research 1, 237-241 (1980) § CALCITONIN AND SOMATOSTATIN ARE LOCALIZED IN DIFFERENT CELLS IN THE CANINE THYROID GLAND 1 i l 2 YOSHISUKE KUSUMOTO Department of Anatomy, Niigata University School of Medicine, Asahimachi 1, Niigata 951, Japan ABSTRACT Localization of calcitonin-like and somatostatin-like immunoreactivity in the puppy thyroid gland was examined by the indirect immunoperoxidase procedure. By the use of the double staining method for calcitonin and somatostatin and of alternate immunostaining in serial, adjacent sections, it has been revealed that calcitonin-like and somatostatin-like immunoreactivity occurs in separate cells. KEY WORDS thyroid gland / parafollicular cells / calcitonin / somatostatin / immunohisto- chemistry The localization of calcitonin in parafollicular stained alternately by indirect immunoperoxi- cells of the mammalian thyroid has been inves- dase procedure using anticalcitonin and antiso- tigated by immunohistochemical techniques (4, matostatin antisera. 10, 19, 26, 27). Since the immunohistochemical 2. The double staining method using 4-Cl- study by Hokfelt and his associates (8), soma- l-naphthol and 3, 3’-diaminobenzidine-tetrahy- tostatin has been reported to occur also in the drochloride (DAB) (20) was applied to single parafollicular cells (23, 28). In the thyroid of sections of 4-5 /am thickness. the rat (22) and the rabbit (3), it has been re- Synthetic human calcitonin and synthetic so- ported that calcitonin and somatostatin occur matostatin (Peptide Institute Protein Research in the same cells. The present paper will report Foundation, Osaka) were used as antigens. An- that these polypeptides are localized in separate ticalcitonin and antisomatostatin sera were cells using the puppy thyroid. Since the dis- raised in rabbits by injection of either antigen. covery of the parafollicular cells (l, 21), the These primary antisera were kindly supplied by thyroid of the dog, especially of young ones, Dr Y. Yamada, Shinrakuen Hospital, Niigata has been known to contain them numerously and Dr S. Ito, First Department of Internal and is favored material also in recent studies Medicine, Niigata University School of Med- (4, 10-15). Yet the relationship between cal- icine, respectively. Anticalcitonin serum dilut- citonin and somatostatin producing cells has ed 100 fold and antisomatostatin serum diluted not been examined in this species as far as we 1,000 fold gave the most favorable reactions. are aware. The specificity of the reactions was checked (1) by adding 10 pg/ml human calcitonin or so- matostatin to the primary antiserum before- MATERIALS AND METHODS hand, (2) by using a 100 fold or 1,000 fold di- In the present study, five puppies of both sexes luted normal rabbit serum instead of the pri- from 1 to about 6 months in age were used. mary antiserum, and (3) by the use of only a Tissue blocks of the thyroid were fixed in Bou- peroxidase-conjugated secondary antiserum. in’s fluid for 16-20 hr and embedded in paraf- fin. Sections, partly cut in continuous series, RESULTS were treated with the following methods: 1. Serial sections of 2 pm thickness were The imrnunoperoxidase method clearly demon- 238 Y. KUSUMOTO 'r,i:.~=:(<.-.>q_ qll 1,- " “in-~ \ . 4;.’_ J -. " "‘ . .1- -L bl,I. ‘Ii-\ ' L ‘X --':;$'.‘ T ..;*h-57I“.4___ -s ,1 '33 '7 _"_‘.i _ a. M) G _ ‘f",1 - 1.6.5in}; i \ {- “ts "1 Vi ~ I '1 l Il- 5-in.__ "Q B Fig. 1 Calcitonin-reactive (A) and somatostatin-reactive (B) cells in the puppy thyroid as dem- onstrated by immunoperoxidase reaction. In micrograph B, a somatostatin-reactive cell ex- tends a cytoplasmic process to interstitial space (arrow). Note that the former cells are by far more numerous than the latter cells. l Calcitonin-reactive cells were distributed widely in the interfollicular and parafollicular areas either in groups or singly and were en- countered constantly and numerously in every section examined (Fig. IA). They were usually oval or polygonal in shape. On the other hand, somatostatin-reactive cells were distributed mostly in the parafol- l licular position. They were much fewer in num- ber than the calcitonin-reactive cells. More- F over, they varied conspicuously in distribution due to the portion of the thyroid cut: they were numerous in the core part of the organ and I Fig. 2 Calcitonin-reactive cells (grey) and somato- I statin-reactive cells (brown) shown by double stain- very scant in the periphery. Somatostatin-re- I ing using 4-Cl-l-naphthol and DAB as substrates active cells were generally polygonal in shape L and tended to be much more angular than the strated in the puppy thyroid that calcitonin-like calcitonin-reactive cells. Some of them extend- immunoreactivity and somatostatin-like immu- ed slender cytoplasmic processes to the inter- noreactivity were localized in specific cells stitial space (Fig. 1B). I which all fall into the category of parafollicular By the use of 4-Cl-1-naphthol for calcitonin cells. 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