ACTA HISTOCHEM. CYTOCHEM. Vol. 3, No. 3, 1970 IMMUNOHISTOCHEMICAL INVESTIGATION OF AMINOPEPTIDASE TOSHIOSUZUKI, KUNIOTAKANO AND KENJIROYASUDA Departmentof Anatomy,School of Medicine,Keio University,Shinjuku, Tokyo Receivedfor PublicationJune 2, 1970 The localization of aminopeptidase was examined on the porcine intestine by use of fluorescent antibody method. Aminopeptidase was present diffusely in the striated border of mucous epithelial cell of jejunum and ileum, supra-nuclear region of epithelial cell, intestinal glandular cells and in goblet cell. The condensed materials in the crypts showed specific fluorescence. The antigen was not always observed in every cell mentioned above, and the distribution pattern of antigen varied according to the stage of digestion. The enzyme was concentrated in the goblet cells of the small intestine but not in those of the large intestine. Brun- ner's gland of duodenum did not contain any aminopeptidase. Though amino- peptidase in kidney and pancreas had immunological common factor with that in the intestine, specific fluorescence was not observed on the tissue sections. For comparative purposes, histochemical staining was carried out by means of Burstone and Folk's method. As a result, the goblet cell of the small intestine did not show any cytochemical reaction, though the specific fluores- cence which showed the site of the enzyme protein was concentrated in it. As far as the striated border of both mucous epithelial cell and intestinal glandular cell is concerned, the results of the cytochemical reaction coincided with those obtained by immunohistochemical study. The reason for the discordance with regard to the distribution of cytochemical reaction and the immuno- fluorescence in the goblet cell remained unknown. There have been very few bistochemical investigation on the localization of exo-and endopeptidases. Burstone and Folk (1956) have demonstrated the location of aminopeptidase on the tissue by using L-leucyl-ƒÀ-naphthylamide and DL-alanyl- naphthylamide as the substrate. From the same purpose, Nachlas et al. (1957, 1960) used L-leucyl-4-methoxy-ƒÀ-naphthylamide as a substrate. Takamatsu (1963) has reported a histochemical method for the demonstration of proteinase, using silver protein as a substrate. More recently, the immunohistochemical study on the localization of trypsinogen and chymotrypsinogen in the acinar cell of porcine pancreas by means of the fluorescenct antibody method, has been made by Yasuda and Coons (1965). Further, by the same technique, pepsin was demonstrated in the bovine stomach (Yasuda, Suzuki and Takano, 1966). Though, a number of attempts for the demonstration of peptidase or proteinase have been tried by many investigators from the early years of the histochemistry, and though there are many excellent techniques for the demonstration of enzymes 130 IMMUNOHISTOCHEMICAL STUDY OF AMINOPEPTIDASE 131 other than protein catabolising enzymes, the histochemistry of protein and protein catabolising enzyme is not yet well developed. This study dealt with the localization of aminopeptidase in the porcine intest- inal mucosa by means of fluorescent antibody technique. In addition, the results were compared with those obtained by the histochemical method developed by Burstone and Folk (1956) for the demonstration of aminopeptidase. MATERIALS AND METHODS The antigen used in this study was crystalline porcine aminopeptidase of a commercial source (Sigma Co.), prepared from the intestinal mucosa. According to the data sheet from the company, 1 unit will liberate 1 ƒÊ mol ƒÀ-naphthylamine per min from L-leucyl-ƒÀ-naphthylamide at pH 7.0 at 37•Ž. 40mg of the antigen dissolved in the phosphate buffered saline was mixed with an equal volume of Freund's complete adjuvant and injected intramuscularly into rabbits. A week after the first injection, 10mg of antigen dissolved in the same solution as mentioned above was mixed with an equal volume of incomplete adjuvant and injected in the same manner. This procedure was repeated four times in order to obtain as high titer antiserum as possible. Five weeks after the first injection, each rabbit recieved an intravenous injection of 10mg of the antigen dissolved in the same buffer as the final booster injection. Six days after the final injection, the animals were bled by heart puncture. The serum was separated and stored in the presence of merthiolate at the concentration of 1: 10,000 at -20•Ž until use. The antiserum contained 70mg/ml of protein and had a titer of 1: 163,840 by the passive hemagglutination test of Stavitsky (1954), using 10mg of antigen. This anti-aminopeptidase serum was conjugated with FITC and purified by the chromatography on Sephadex G-25 (Yasuda et al., 1963, 1964, 1965) and DEAE cellulose columns according to the technique of McDevitt et al. (1963). The molar F/P ratio (McDevitt et al., 1963) of the conjugated antibody solution was 0.92. Precipitin reactions were carried out on the Ouchterlony plate using 0.8 per cent Noble agar (Difco Co.). Consequently, two precipitin bands were encountered between antigen and antibody (Figl 1). The frozen sections of porcine intestine were cut at 4ƒÊ thick in a cryostat at -18•Ž . The other blocks were fixed with several kinds of fixatives at 0•Ž, de- hydrated with ethanol and embedded in paraffin. The fixatives employed in this study were twenty-two kinds. Among these fixatives, 1 per cent glacial acetic acid in 99 per cent ethanol was the most satisfactory for the preservation of the antigen. The hematoxylin-eosin staining, PAS staining and toluidine blue staining were carried out to observe the histological detail of the porcine intestine. The histo- chemical method to demonstrate aminopeptidase (Burstone and Folk, 1956) was also applied to the tissue to compare with the immunohistochemical results. RESULTS In general, the histological pattern of porcine small intestine is rather similar to that of cat and dog than that of man. Goblet cells are abundant, while the Paneth cells are very scanty or almost lacking. Furthermore, the most characteristic feature 132 SUZUKI, TAKANO AND YASUDA IMMUNOHISTOCHEMICAL STUDY OF AMINOPEPTIDASE 133 of pig small intestine is that the crypt of Lieberkuhn is much longer than that of man and other higher vertebrates. Duodenum: The epithelial cells covering the free surface of the villi are simple columnar. The wall of the crypt of Lieberkuhn is lined with a low columnar epithelial cells. The striated border is noticed at the free surface of the epithelial cells of the mucous membrane and of cells covering the crypt of Lieberkiihn. The few goblet cells are irregularly scattered among the mucous epithelial cells and intestinal glandular cells. The observation by fluorescence microscope revealed the concentration of antigen in the goblet cells. But, the specific fluorescence is not encountered in both mucous epithelial cells and the intestinal glandular cells. The striated border of the mucous epithelial cells and intestinal glandular cells is occasionally bright. Es- pecially, the fluorescence recognized on the striated border of the cells lining the crypts of Lieberkuhn is intensely bright. The fluorescenct materials are not seen in lamina muscularis mucosae, smooth muscle fibers, and in blood cells. Jejunum and ileum: The histological features differ much from those of the duodenum except for the presence of Brunner's gland. The intestinal crypt is much deeper than that of the duodenum. Goblet cells are much more abundant than in duodenum. The abundance of goblet cells resembles to the histological pattern of human colon. Bright green fluorescence is observed in the striated border and in the cytoplasm of the supra-nuclear region of each goblet cells and mucous epithelial cell (Figs. 2,3, and 4). The cells which line along the crypt are filled with fluorescent material. The apical portion of cytoplasm of Paneth cell-like cells situated at the bottom of the intestinal crypt is also fluorescent (Fig. 2). The masses of secretory products which are projecting into the crypt and lumen of the intestinal gland show brilliant flu- orescence (Fig. 3). The nucleus of every cell shows the negative image. Large intestine: Goblet cell, mucous epithelial cell, intestinal glandular cell and striated border of these cells are quite free of antigen. Fig. 1. Precipitin reaction on the agar plate (Ouchterlony technique). Large center cup contained anti-aminopeptidase serum, surrounding cups are filled with different dilutions of the antigen, con- taining 20mg/ml, 10mg/ml, 5mg/ml and 2.5mg/ml respectively clockwise from the upper left cup. Two precipitin lines are found between antigen and antiserum. Fig. 2. The cross and longitudinal section of the intestinal gland of the porcine ileum. In the intestinal crypt, diffuse brilliant specific fluorescence is observed in the goblet cells. And intense fluorescence is also found in cytoplasm of the cells lining along the crypt. On the upside of this figure, the cross section of secretory portion of the gland of Lieberkiihn is observed. Besides the findings that specific fluorescence is found in goblet cells, Paneth cell-like cell (P) is stained with labeled anti-aminopeptidase serum. The lumen of the intestinal gland is partly filled with the material which shows the bright specific fluorescence. •~250. Fig. 3. The crypt of porcine intestinal gland is shown in this picture. The secretory product excreted from goblet cells shows the bright specific fluorescence, which is going to spread over the striated border. •~400. Fig. 4. The crypt of porcine intestinal gland. Specific fluorescence is observed in goblet cells and along the striated border of crypt cells. •~400. 134 SUZUKI, TAKANO AND YASUDA Heterogeneous organ: As an organ which has aminopeptidase, the porcine kidney was selected and stained with labeled gamma-globulin solution. As a result, only autofluorescence was seen in the wall of blood vessels, but no specific fluorescence is seen in the kidney. Further, the results obtained by this method was compared with those by histo- chemical technique developed by Burstone and Folk.