Acta histochem. 87, 95-97 (1989) VEB Gustav Fischer Verlag Jena

Sektion Biologie, Wilhelm-Pieck-Universitat Rostock, GDR

Histochemical localization of a-amylase in the pancreas of pike (Esox lucius) by indirect immunofluorescence

By EDDA SIEGL, MATTHIAS QUOSIGK, and HARALD PLANTIKOW

With 2 Figures

(Received March 16, 1989; accepted May 17, 1989)

Summary

IX-amylase is an enzyme, rarely detected histochemically. Only a few examples are known in mammals. A method is described for the histochemical detection of an IX-amylase in the pancreas of pike using a rabbit antiserum against pike amylase and FITC-labelled anti-rabbit immunoglobulin.

1. Introduction

For studies of the digestive physiology of fish, it is necessary to investigate the exocrine pancreas. As this tissue is diffusely distributed in many fish species, it is not easily to localize and its investigation is rather difficult. Immunhistochemical methods could be helpful especially for localization of amylase storage and transport in the cell. Amylase is histochemically detected only in a few cases and only in mammals. A starch film method was used. This method detects fields with amylase activity by means of a thin layer of starch agar below the tissue section. The demonstration of not degraded starch molecules was done with iodine staining'(SHEAR and PEARSE 1963) or PAS­staining (TREMBLAY and TEASDALE 1964). Both methods show disadvantages, because they do not show the localization of amylase in the cell. A direct immunofluorescence technique with FITC­labelled anti-amylase serum was described in pig pancreas (YASUDA 1964). We used an indirect immunofluorescence technique with the application of antibodies from rabbit against pike amylase and FITC-labelled anti-rabbit immunoglobulin for the detection of amylase in pike pancreas.

2. Material and methods

Extraction of amylase: Pancreatic tissue of pike was homogenized with isotonic N aCI-solution in a proportion of 1: 10. The extraction was done according to a method of SCHRAMM and LOYTER (1977) based on the formation and separation of a glycogen-amylase complex.

Immunization: 4 month old rabbits were immunized with the glycogen-amylase complex. The glycogen component is not immunogen. Between the 1st and the 2nd injection (s.c.) with each 4 mg (86!lg Protein/mg) with KFA (complete Freund's adjuvans), there was a break of 6 weeks. The rabbit were immunized in the following 6 weeks 3 times/week with 4 mg first, then, 8, 10, and 20 mg without KFA s.c. in the leg.

The formation of antibodies was tested with Ouchterlony assay. The rabbits were bled, and serum was obtained. Indirect immunofluorescence technique (STORCH 1986): Immediately after removing the pancreatic tissue, it was

frozen in a dry-ice-aceton mixture. Sections (10 !lm) were cut by a cryostat, mounted on protein-glycerin coated slides, air-dried for 5 d (4 QC), and fixed in ice-cold aceton. After washing in PBS (Phosphate buffered saline) pH = 7.0, the sections were incubated with anti-amylase serum (diluted 1 : 20) for 48 h (4 QC) in a moist chamber.

The sections were washed again and incubated with FITC-labelled anti-rabbit Ig (SIFIM Berlin, DDR) for 1 h.

7*

96 E. SIEGL, M. QUOSIGK , and H. PLANTIKOW

Fig. I. Ouchterlony assay with pike amylase (in the outer wells) and antiserum against pike amylase (in the central well) . The highest concentration of antigen (undiluted, 20 mg/ml) at the top, the lowest (1: 32) on the left. The antiserum is undiluted.

Fig. 2. Indirect immunofluorescent localization of amylase in the pancreas of pike with anti-pike amylase serum from the rabbit and FITC-anti-rabbit immunoglobulin. Note the strong fluorescence in the acinus cells around the blood vessel and on the edge of the acini.

Histochemical localization of <x-amylase 97

After washing with PBS, the sections were inspected with fluorescence microscope FLUOVAL 2® (VEB Carl Zeiss lena, DDR) with epiillumination, filtercombination B 229 g and KP 490.

Controls: (I) Incubation of pancreas sections with FITC-anti-rabbit serum only. (2) Incubation of muscle tissue sections with both antisera.

3. Results

In preliminary experiments, an antiserum against commercial pig amylase was produced supposing to detect amylase in pike pancreas with this antiserum. However, cross reactions between anti-pig-amylase serum and pike amylase were not observed. Thus it was necessary to extract amylase from pike pancreas to produce a specific antiserum against this antigen. The protein content of extracted ex-amylase was low (86 !tmlmg), due to the high glycogen content. The separation of glycogen would have required high methodical effort. Since glycogen is not immunogen, separation was omitted.

Nevertheless, a satisfactory antibody titer was obtained with the immunization schema described. Only one precipitation line was observed with the antigen pike amylase (Fig. 1).

The histological demonstration of amylase with indirect immunofluorescence gave good results. Controls (see material and methods) show no unspecific fluorescence. A high amylase content was detected especially in acinus cells around blood vessels (Fig. 2). A granulated fluorescence concentrated to the edge could be observed in the acini.

4. Discussion

A similar distribution of amylase activity as in our investigations but with starch film method and iodine staining, was found in the pancreas of rats (SHEAR and PEARSE 1963). The PAS-staining also used shows unspecifities in some cases (TREMBLAY and TEASDALE 1964) because parts of tissue without amylase activity were stained (SHEAR and PEARSE 1963). Use of direct FITC-Iabelled anti-amylase serum for detection of amylase in pig pancreas resulted in a fine granulation near the cell basis similar to our observations (YASUDA 1964).

Comparable results in fish pancreas are not known.

References

SCHRAMM, M., and LOYTER, A., Purification of <x-amylase by precipitation of amylase-glycogen-complexes. Methods Enzymol. 8, 533-537 (1966).

SHEAR, M., and PEARSE, A. G. E., A starch substrate film method for the histochemical localization of amylase. Exp. Cell Res. 32, 174-176 (1963).

STORCH, W., lmmunfluorescenz. In: LUPPA, H., AMBROSIUS, H., und STORCH, W. (eds.), Handbuch der Histochemie 412. Fischer, Stuttgart/New York 1986, pp. 189-296.

TREMBLAY, G., and TEASDALE, F., Histochemical study of amylase activity in the embryonic rat pancreas. Exp. Cell. Res. 35, 211-212 (1964).

YASUDA, K., Immunocytochemical demonstration of amylase in the acinar cells of the pig pancreas. II. Intemationaler KongreB fUr Histo- und Cytochemie, Frankfurt/Main, Springer, BerlinlGottingenlHeidelberg 1964, p. 153.

Authors' address: Doz. Dr. sc. nat. EDDA SIEGL, Wilhelm-Pieck-Universitiit Rostock, Sektion Biologie, WB Tierphysiologie, Universitiitsplatz 2, Rostock 1, DDR - 2500.