Bodian's silver impregnation of endocrine cells

Summary We have recently shown that a variety of proteins, including albumin and immunoglobulins conjugated to colloidal gold, strongly binds to certain basic peptide sequences, and neurohormonal peptides. Silver proteinate, used in the classical Bodian's neurohistological procedure, is now shown to bind to the same peptide sequences in cytochemical model systems. In tissue, gastrin cells and glucagon cells have been reported to show strong unspecific immunocytochemical staining and these cell types also stain in the Bodian's procedure. These results suggest that certain types of unspecific immunocytochemical staining and the Bodian's silver staining method may depend upon a common mechanism, involving binding of labelled or aggregated protein to basic and hydrophobic sequences in tissue.     

Application of silver stains to cytologic specimens of neuroendocrine tumors metastatic to the liver

Cytologic specimens of neuroendocrine tumors metastatic to the liver were examined with regard to their silver staining properties after the application of argentaffin and argyrophil staining techniques (Masson, Grimelius and Sevier-Munger). In tumors with a content of serotonin (small intestine carcinoids), the presence of this substance was demonstrated cytologically as an argentaffin reaction in individual tumor cells; however, formalin fixation was a prerequisite for positive staining. Melanin in malignant melanoma cells displayed a positive argentaffin reaction, irrespective of the fixation used (air drying, formalin, Bouin's fluid or acetone-alcohol). Thus, serotonin and melanin can be distinguished in cytologic samples of neuroendocrine tumors by the use of the Masson argentaffin reaction with different fixatives. The nonargentaffin-positive neuroendocrine tumor cells were weakly stained or unreactive with the Grimelius argyrophil technique. The Sevier-Munger argyrophil technique was negative or gave a disturbing nonspecific background staining reaction that was difficult to interpret in the cytologic samples. Thus, the Grimelius method appears to be the most useful silver stain for identifying neuroendocrine tumor cells in cytologic material, irrespective of their hormone content, since both argentaffin-positive and argentaffin-negative cell samples were stained at least to some degree.     

Silver stains in the study of endocrine cells of the gut and pancreas

The usefulness of argentaffin (Masson) and argyrophil (Davenport, Sevier-Munger and Grimelius) staining methods for identification of endocrine cell types in the gastrointestinal tract and pancreas is discussed and comments are made on the techniques themselves. The applicability of silver impregnation methods in the histopathological investigations of endocrine tumours in the above-mentioned organs is outlined, and the chemical background of the silver reactions is briefly reviewed.     

Nutrient sensing receptors in gastric endocrine cells

Sensing protein breakdown products in the luminal content is of particular importance for the regulation of digestive activities in the stomach which are mainly governed by gastric hormones. The molecular basis for tuning the release of hormones according to the protein content is still elusive. In this study we have analysed the murine stomach for candidate nutrient receptors. As a promising candidate we have concentrated on the broadly tuned amino acid receptor GPRC6A. Expression of GPRC6A could be demonstrated in different regions of the murine stomach; especially in the gastric antrum. Using immunohistochemical approaches, a large cell population of GPRC6A-positive cells was visualized in the basal half of the antral gastric mucosa. Molecular phenotyping of GPRC6A-immunoreactive cells revealed that most of them contained the peptide hormone gastrin. A small population turned out to be immunoreactive for somatostatin. In search for additional amino acid receptors in antral gastric mucosa, we obtained evidence for expression of the gustatory amino acid receptor subunit T1R3 and the calcium-sensing receptor CaSR. Many CaSR-cells were found in the gastric antrum and most of them also contained gastrin; very similar to GPRC6A-cells. In contrast, T1R3 was found only in a small population of gastrin-negative cells. The finding that GPRC6A-and CaSR-receptors are both expressed in many if not all gastrin cells strongly suggests that both receptor types are co-expressed in the same cells, where they could form heterodimers providing a unique response spectrum of these cells.     

The application of lipid-soluble stains in plastic-embedded sections

Summary The present study was designed to develop a toutine method for direct demonstration and precize localisation of lipid substances in tissue sections. A panel of lipid-rich tissues was fixed in 4% buffered formaldehyde, infiltrated, and embedded in the water-soluble plastics Technovit 7100, EFL-67, and JB-4. The use of alcohol containing fluids was avoided. Staining with the lipid-soluble dyes Sudan Black B and Oil Red O revealed excellent preservation of tissue lipids in Technovit 7100 embedded sections when compared with cryostat sections of the same tissue specimens. Lipid preservation in EFL-67 and JB-4 embedded sections was inconsistent, even when infiltration and polymerization procedures were performed at 4°C. Combination of lipid-soluble dyes with the periodic acid Schiff Jones' methenamine silver, or Gomori' reticulin method allowed for an exact localization of lipids in high-quality Technovit 7100 embedded sections. The procedure herein is casily applicable in routine histopathology practice.     

The application of lipid-soluble stains in plastic-embedded sections

The present study was designed to develop a routine method for direct demonstration and precise localization of lipid substances in tissue sections. A panel of lipid-rich tissues was fixed in 4% buffered formaldehyde, infiltrated, and embedded in the water-soluble plastics Technovit 7100, EFL-67, and JB-4. The use of alcohol containing fluids was avoided. Staining with the lipid-soluble dyes Sudan Black B and Oil Red O revealed excellent preservation of tissue lipids in Technovit 7100 embedded sections when compared with cryostat sections of the same tissue specimens. Lipid preservation in EFL-67 and JB-4 embedded sections was inconsistent, even when infiltration and polymerization procedures were performed at 4 degrees C. Combination of lipid-soluble dyes with the periodic acid Schiff, Jones' methenamine silver, or Gomori' reticulin method allowed for an exact localization of lipids in high-quality Technovit 7100 embedded sections. The procedure herein is easily applicable in routine histopathology practice.     

Neuron-specific enolase in mucosal endocrine cells and carcinoid tumours of the small intestine: A comparative study with neuron-specific enolase immunocytochemistry and silver stains

Summary Endocrine cells of human small intestinal mucosa, small intestinal carcinoids and carcinoid liver metastases were stained with an immunocytochemical technique using an antiserum against neuron-specific enolase (NSE), with the argyrophil technique of Grimelius and with the argentaffin technique of Masson. In the normal mucosa, scattered NSE-immunoreactive cells were seen mainly in the deeper parts of the crypts. These cells, as shown in the same sections, corresponded to the argentaffin and/or argyrophil cells indicating that they were of endocrine type.All intestinal carcinoids (16 cases) displayed NSE immunoreactivity. However, this reaction did not correlate on the cellular level with the silver techniques employed. Thus, many tumour cells were NSE immunoreactive but lacked an argentaffin or argyrophil reaction and vice versa. On the light microscopical level the silver techniques reveal the presence of neurohormonal granules in the tumour cells, while the NSE immunoreactivity appears to disclose neuroendocrine differentiation of the tumour cells irrespective of their hormone and granular content.Out of 13 carcinoid liver metastases, eight displayed strong NSE immunoreactivity, three were weakly stained and two were unreactive. Consecutive or the same tumour sections showed an argentaffin and argyrophil reaction in all carcinoid metastases. Since silver staining provides one type of information and NSE immunocytochemistry another, they provide in combination a good discriminator for neuroendocrine tumours.     

Ultrastructural characteristics of endocrine and glandular gastric cells

Electron microscopy of human gastric mucosa has demonstrated that the endocrine cells are closely and specifically related to the adjacent glandular cells and the basal membrane. Cytoplasmic strands of the adjacent cells surround the endocrine cells and their projections, probably, regulating their secretion. The above outlined relations are considered a structural basis for complex regulatory endocrine and paracrine functions of the gastric endocrine cells.     

Influence of autolysis on rat gastric endocrine cells

Summary In the present study histochemical parameters of the rat gastric endocrine cells were followed up in the course of 24-h autolysis, and their ultrastructure was studied during autolysis lasting for 60 min. The autolysis occurred at 37°C.In the light microscope, with the histochemical methods applied, only EC, ECL and G cells could be identified during the one-hour autolysis. With the autolysis proceeding for 6 and 12 h, only argyrophil method according to Grimelius (1968) enabled visualization of gastric argyrophilic cells. After 24 h of autolysis, none of the methods applied (not even the Grimelius method) proved to be adequate for successful demonstration of the gastric endocrine cells.In the course of 60-min autolysis, electron microscopic examination provided identification of the EC, ECL, AL, D₁, and G cells with the characteristical ultrastructural appearance of granules. The granules of the endocrine cells (G cells included) were found to be considerably resistant to autolysis. The effect of 60-min autolysis did not induce granule emiocytosis or dissolution of granule content. Autolysis exceeding five minutes resulted in damage of the mitochondria of different degrees and in dilatation of the profiles of endoplasmic reticulum (particularly in G and AL cells).The results obtained in the present study demonstrate the feasibility of in vitro experimental stimulation since the endocrine granules have proved to be resistant to the effects of simultaneously developing autolysis.     

Evolution of the silver and gold stains in neurohistology

Scientific investigations depend on the reliability of the observations that can be made. This reliability is determined in part by the understanding of the techniques and technology used to make the observations. The limitations and the strengths of the methodology and the equipment used must be evaluated thoroughly. The extent to which this is and has been the case for the use of the metal based stains in neuroscience is the subject of this paper. I evaluate the metallic stains used for neuroscience from several perspectives. I review briefly the state of neurohistology prior to its “golden years,” 1870-1910. Then I trace the development of the silver based stains used for neurohistology. I wanted to discuss the reasoning used by the originators of the silver based techniques in developing their specific procedures, but discovered that while procedures may be published, the methods and ideas used to arrive at the final procedures are not usually described in published work.     

Evolution of the silver and gold stains in neurohistology

Scientific investigations depend on the reliability of the observations that can be made. This reliability is determined in part by the understanding of the techniques and technology used to make the observations. The limitations and the strengths of the methodology and the equipment used must be evaluated thoroughly. The extent to which this is and has been the case for the use of the metal based stains in neuroscience is the subject of this paper. I evaluate the metallic stains used for neuroscience from several perspectives. I review briefly the state of neurohistology prior to its “golden years,” 1870–1910. Then I trace the development of the silver based stains used for neurohistology. I wanted to discuss the reasoning used by the originators of the silver based techniques in developing their specific procedures, but discovered that while procedures may be published, the methods and ideas used to arrive at the final procedures are not usually described in published work.     

Bodian's silver impregnation of endocrine cells. A tentative explanation to the staining mechanism

We have recently shown that a variety of proteins, including albumin and immunoglobulins conjugated to colloidal gold, strongly binds to certain basic peptide sequences, and neurohormonal peptides. Silver proteinate, used in the classical Bodian's neurohistological procedure, is now shown to bind to the same peptide sequences in cytochemical model systems. In tissue, gastrin cells and glucagon cells have been reported to show strong unspecific immunocytochemical staining and these cell types also stain in the Bodian's procedure. These results suggest that certain types of unspecific immunocytochemical staining and the Bodian's silver staining method may depend upon a common mechanism, involving binding of labelled or aggregated protein to basic and hydrophobic sequences in tissue.     

Application of the thiocarbohydrazide method for vicinal glycol group detection to the study of gastric mucosa endocrine cells

Summary The thiocarbohydrazide-silver proteinate (TCH-SP) method was applied to the study of cat, rabbit and mouse gastric mucosa endocrine cells. After 24-h treatment with thiocarbohydrazide (TCH), glycogen was seen in the hyaloplasm of X, D, P, A and O cells but not in EC, EC-like or D₁ cells. With flotation times as short as 30 to 40 min glycogen was readily detected in X cells. Secretory granules of EC cells were constantly stained, while those of D₁ cells failed to react. In most experiments granules of X, A and O cells showed peripheral staining, while in others staining of variable intensity affected the entire granular cross-section in X, D and P cells. With 72-h exposure to TCH, EC and EC-like cells showed particles resembling glycogen, even staining or only peripheral staining of certain EC cell granules. From the results of this and previous studies, EC cell staining is believed to be due wholly or partly, according to exposure times, to the action of silver proteinate, while that of certain non-EC cells is probably a specific indicator of complexed carbohydrates.     

Evolution of the silver and gold stains in neurohistology.

Scientific investigations depend on the reliability of the observations that can be made. This reliability is determined in part by the understanding of the techniques and technology used to make the observations. The limitations and the strengths of the methodology and the equipment used must be evaluated thoroughly. The extent to which this is and has been the case for the use of the metal based stains in neuroscience is the subject of this paper. I evaluate the metallic stains used for neuroscience from several perspectives. I review briefly the state of neurohistology prior to its "golden years," 1870-1910. Then I trace the development of the silver based stains used for neurohistology. I wanted to discuss the reasoning used by the originators of the silver based techniques in developing their specific procedures, but discovered that while procedures may be published, the methods and ideas used to arrive at the final procedures are not usually described in published work.     

Localization of chromogranin A-immunoreactivity in bovine gastrointestinal endocrine cells with special reference to Grimelius silver stain.

Immunohistochemical studies of the gastrointestinal tract were carried out to characterize the cells exhibiting immunoreactivity for chromogranin A (CGA), a glycosylated protein primarily found in secretory granules of the adrenal medulla. Double immunostaining for gastrointestinal hormones and CGA revealed that in the bovine gastrointestinal tract CGA immunoreactivity occurs in mucosal epithelial cells containing gastrin, glucagon, substance P or motilin, but not in those containing somatostatin. Combined staining with anti-CGA serum and Grimelius' silver demonstrated frequent association of the two stains in a variety of endocrine cells. However, intracellular distribution of the two stains was different: CGA-immunoreactivity was detected in both supra- and infranuclear cytoplasm, whereas Grimelius' silver was mostly localized in the infranuclear region. These results suggest that CGA is the target of Grimelius' silver, as postulated recently (Rindi et al., 1986), but that some subcellular structure-related modification of molecules such as sialation is necessary for the positive Grimelius reaction.     

Proliferative activity of gastric and duodenal endocrine cells in the rat

The replicative activity and migration of gastrin, somatostatin and serotonin cells in rat stomach and duodenum was studied using combined immunocytochemistry and autoradiography after 3H thymidine pulse-labeling. Our results show that a small proportion of gastrin, somatostatin and serotonin immunoreactive cells displays proliferative activity. The overall labeling index ranged from 1.3% for gastric endocrine cells to 3.2% for duodenal endocrine cells. In a pulse chase experiment, labeling indices of immunoreactive cells were estimated at several time intervals after 3H thymidine administration. Significant differences in labeling index were not found. Migration of 3H thymidine labeled endocrine cells towards the luminal surface was not found in the stomach nor in the duodenum. It is concluded that 1) these endocrine cells have replicating activity; 2) the replicative activity of endocrine cells is higher in the duodenum than in the stomach; 3) the various cell types do not show significant differences in replicating activity and 4) endocrine cells did not seem to migrate to the luminal surface of the mucosa along with the other epithelial cells.     

Proliferative activity of gastric and duodenal endocrine cells in the rat

Summary The replicative activity and migration of gastrin, somatostatin and serotonin cells in rat stomach and doudenum was studied using combined immunocytochemistry and autoradiography after ³H thymidine pulse-labeling. Our results show that a small proportion of gastrin, somatostatin and serotonin immunoreactive cells displays proliferative activity. The overall labeling index ranged from 1.3% for gastric endocrine cells to 3.2% for duodenal endocrine cells.In a pulse chase experiment, labeling indices of immunoreactive cells were estimated at several time intervals after ³H thymidine administration. Significant differences in labeling index were not found. Migration of ³H thymidine labeled endocrine cells towards the luminal surface was not found in the stomach nor in the doudenum.It is concluded that 1) these endocrine cells have replicating activity; 2) the replicative activity of endocrine cells is higher in the duodenum than in the stomach; 3) the various cell types do not show significant differences in replicating activity and 4) endocrine cells did not seem to migrate to the luminal surface of the mucosa along with the other epithelial cells.     

Preliminary evaluation of gastric endocrine cells in rats with experimental uremia

Chronic renal failure can be the cause of various disturbances in hormonal and electrolyte metabolism, including calcium and phosphate metabolism. The aim of this study was the evaluation of pyloric endocrine cells in Wistar rats with experimental uremia. Fragments of gastric pylorus were collected 30 days after nephrectomy. Paraffin embedded sections were stained with H+E and by silver impregnation. We also performed immunohistochemical reactions with the use of specific antibodies against calcitonin gene-related peptide (CGRP), synaptophysin (SY), somatostatin (ST), and neuronal specific enolase (NSE). The rats with experimental uremia showed an increase in the number of endocrine cells and in intensity of all the examined reactions.