Immunoelectron microscopic observation of chicken glucagon-like peptide (GLP)-1-containing cells in tissues derived from thin section, paraffin block and conventional method

Abstract

The purpose of the present study was to investigate the possibility of immunoelectron microscopic observation of endocrine cells in paraffin-embedded tissues. The procedure, which involves reprocessing from sliced tissues and immunohistochemical staining by colloidal-gold immunolabeling of paraffin sections from paraffin blocks, was able to reveal the fine characteristics of secretory granules containing glucagon-like peptide-1. Morphometric analyses of the secretory granules showed no significant differences between the reprocessing procedure and a conventional post-embedding procedure, which was performed as a control. The reprocessing procedure has some advantages besides providing information on secretory granules containing the amino acid peptide. For example, the same cell can be observed under both a light microscope and the electron microscope. In addition, the high-electron densities of silver-enhanced gold particles are easily recognized, and the boundary between the profile of the granules and the immunogold labeling is clearly shown at the electron microscopic level. Furthermore, the procedure, which is inexpensive and does not require special devices, can effectively use precious samples that are already paraffin-embedded and unable to be obtained twice, such as the case for endangered animals and rare pathological tissues. To the best of our knowledge, the present study is the first to report the advantages of the reprocessing method for sliced paraffin sections of gut endocrine cells.

Fig. 1.

Immunohistochemical findings of GLP-1-positive cells in the chicken ileum. (a) A section stained with the PrLSB procedure and counterstained with Mayer’s hematoxylin. (b) A section stained with the PrCG procedure. The immunoreactive cells are distributed in the crypts and lower part of the villous epithelium (arrows). Bars=200 µm.

Fig. 2.

Summary of the PrCG procedure from a paraffin block to electron microscopic observation. (a) Distal ileum embedded in paraffin and paraffin sections mounted on a glass slide. (b) A section stained with the PrCG procedure examined under a light microscope without a coverslip. The arrow indicates an immunoreactive cell observed under the electron microscope in panel d. (c) Dissected sections including immunoreactive cells embedded in Quetol 812. The left block was detached from the glass slide by warming. (d) Electron micrograph of an immunoreactive cell. The cell surrounded by a dashed line is the same cell indicated by the arrow in panel b. Secretory granules accumulate in the basal cytoplasm of L-cells (asterisk). Bar=5 µm.

Fig. 3.

Low-magnification electron micrographs of the chicken distal ileum stained with the PrLSB (a) and PrCG (b) procedures. GLP-1-positive cells are easily identified at low magnification by the dark DAB reaction products and gold particles enhanced by silver reactions (arrows). Degradation of the nucleoplasm and cytoplasm is observed in both procedures. An enterochromaffin cell is observed adjacent to the GLP-1-positive cell in panel (b). Bars=2 µm.

Fig. 4.

Electron micrographs of GLP-1-positive cells in the chicken distal ileum stained with the PrLSB (a), PrCG (b) and PoCG (c) procedures. The cells contain round-to-oval secretory granules without a halo. The electron densities of the secretory granules observed with the three different experimental methods are medium and almost the same (arrows). The secretory granules of an enterochromaffin cell adjacent to the GLP-1-positive cell show polymorphous shapes and high-electron density (asterisk) (b). The post-embedding procedure shows fine preservation of the nucleoplasm and cytoplasm (c). Bars=1 µm. (d) High-magnification view of the secretory granules in panel (a). The DAB reaction products precipitate around the secretory granules (arrows) and in the cytoplasm (arrowheads). The profile of the granules is ambiguous. (e) High-magnification view of the secretory granules in panel (b). The particles of enhanced colloidal gold mainly precipitate around the secretory granules (arrows), and a few of them are located in the cytoplasm (arrowheads). The profile of the secretory granules is clearly observed. (f) High-magnification view of the secretory granules in panel (c). Almost all of the particles of colloidal gold are diffusely arranged on the secretory granules (arrows), and very few of them are located in the cytoplasm (arrowheads). The profile of the secretory granules is very clearly observed. Bars=200 nm.