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.     

Antipeptide antibodies differentiate between long and short isoforms of the D2 dopamine receptor.

We have developed specific antibodies directed against two synthetic peptides corresponding to defined sequences in the D2 dopamine receptor. One peptide is from a region that is present only in the 'long' isoform of the receptor, whereas the other is from a region that is common to both. These antibodies are able to recognize the native receptor as judged by immunocytochemical staining of cells transfected with dopamine receptor DNA. One antibody was shown to be specific for the 'long' form of the receptor and reacts only with cells transfected with the 'long' DNA subtype and not with those transfected with the 'short' DNA subtype. This recognition is specific and can be inhibited by the corresponding free peptide and not by a non-relevant peptide.     

Argyrophilic nucleolar organizer regions

Silver staining techniques developed to demonstrate argyrophilic nucleolar organizer regions (Ag-NORs) have been widely applied in a variety of cell kinetic studies, using the mean number of AgNORs in tumour cells as a marker for malignancy of certain types of neoplasms. However, the AgNOR techniques currently available are not entirely satisfactory, as unspecific silver precipitates readily form in the sections. On the other hand, the contrast staining, may be so weak as to render identification of the AgNORs difficult. In the present study, some of the key factors influencing the outcome of AgNOR staining were evaluated in a more systematic way. A modified AgNOR staining procedure is now proposed, giving highly contrasting AgNORs with minimal unspecific silver precipitation, thus facilitating both manual and computerized counting. The new technique involves the use of microwave irradiation in order to shorten the processing time, the use of gelatin as a protective colloid, and a Farmer's solution to optimize the specificity of the technique.     

核仁组成区相关嗜银蛋白染色革新法

核仁组成区相关嗜银蛋白（ＡｇＮＯＲ）银染技术已广泛用以研究细胞生长活性,根据ＡｇＮＯＲ数目多少来判定肿瘤的良恶性和对肿瘤进行鉴别诊断。然而ＡｇＮＯＲ技术至今还存在背景非特异性银颗粒沉积问题而影响结果判定。本研究发现影响背景染色结果的主要因素是明胶的质量,采用Ｆａｒｍｅｒ’ｓ液可以清除背景染色,运用微波炉染色不仅可以缩短染色时间而且可以减少银用量。     

Immunocytochemistry: its evolution and criteria for its application in the study of epon-embedded cells and tissue

The word immunocytochemistry is currently used to describe a number of methods that can be employed to localize antigens within cells by means of antigen-specific antibodies. In this article we will review a number of these methods, including immunofluorescence, immunoperoxidase, avidin-biotin, and colloidal-gold techniques. The advantages and disadvantages of the various methods are discussed, special attention being focused upon immunocytochemical staining of plastic-embedded tissue. Studies on the light microscope level show that embedding tissue in plastic prior to immunoperoxidase staining not only improves visualization of antigen-specific staining but also provides an accurate and efficient means of prescreening tissue for antigen prior to immunocytochemical staining on the electron microscope level. Varying section thickness between 1 and 3 microns does not significantly influence staining, whereas the fixative used to preserve the tissue under study does. On the electron microscope level, the colloidal gold technique appears superior to immunoperoxidase staining. It is both esthetically more pleasing and highly sensitive. Of five different colloidal gold methods tested, the most sensitive is the two-step technique that employs an antigen-specific primary antibody followed by a gold-labeled secondary antibody. Throughout this article, special emphasis is placed on the use of proper controls, both on the light and electron microscope levels. Where possible, such controls should include substitution of specific antiserum with normal serum; the use of antigen-adsorbed antiserum; the use of antisera with specificities for antigens not present in the tissue being studied; the use of tissue previously shown to be stainable for the antigen; and if cultured cells are being studied, the use of a number of cell types that do not contain the antigen.     

Nonspecific immunocytochemical reactions with certain neurohormonal peptides and basic peptide sequences

Immunocytochemical staining experiments on filter paper or nitrocellulose models reveal that many, but not all, neurohormonal peptides, as well as poly-L-lysine, strongly bind a number of labeled reporter molecules, including colloidal gold- or peroxidase-labeled IgG, protein A, streptavidin, and albumin. Peptides displaying this type of (nonspecific) binding are basic; they include ACTH, VIP, opioid peptides, and poly-L-lysine. Pre-absorption of labeled probes with excess ACTH[1-24] or poly-L-lysine abolishes or greatly reduces binding not only to the homologous but also to the heterologous peptides tested. A number of cell types previously reported to display nonspecific immunoglobulin binding contain one or several of the basic neurohormonal peptides shown to display nonspecific absorption of labeled IgG, protein A, streptavidin, and albumin. This nonspecific absorption is reversed neither by high salt nor high pH conditions, nor by a number of detergents and blocking proteins. One dynorphin antiserum also displays nonspecific binding to the peptides as well as to pancreatic glucagon cells, and this nonspecific staining can be blocked by basic peptide pre-absorption (whether homologous or heterologous). These results suggest a need for caution when immunocytochemical studies of a number of basic polypeptides are interpreted, and also suggest the inclusion of novel control procedures in immunocytochemistry.     

Effect of Temperature on Argyrophil Impregnation: Development of A High Temperature Rapid Argyrophil Procedure

Out studies on the effects of temperature on the demonstration of neurosecretory granules using argyrophil stains indicate an inverse relationship between the time needed for staining and temperature of the silver and reducing solutions. Careful monitoring of the temperature of silver solutions during the Grimelius procedure and its modifications show long incubation times serve in large part only to bring the solutions to reaction temperature. Tissue sections added when this temperature has been reached will stain with the same intensity as sections impregnated for the entire incubation period. We have modified the argyrophil procedure so that double-impregnation with solutions preheated to 60-70 C and development in Bodian's reducer prepared with preheated water rapidly demonstrates secretory granules. Our method does not require a microwave oven and much shorter incubation periods are required than with usual procedures. It is not necessary to incubate sections in hot solutions for extended periods of time, which can lead to detachment of sections, nonspecific staining and decomposition of the silver solution. Rinsing after impregnation and before development greatly increases contrast of argyrophil cells by reducing background staining. Our procedure results in more reliable staining of argyrophil and argentaffin cells and takes only ten minutes.     

Staining Senile Plaques using Bodian's Method Modified with Methenamine

A new method is presented for staining various types of senile plaques isolated from the brains of patients with Alzheimer type dementia and related diseases in paraffin embedded sections using a modified Bodian's method with methenamine. This methenamine-Bodian method made it possible to observe diffuse plaques and other amyloid deposits which are barely detected by Bodian's original method. The staining of senile plaques by the method presented here was comparable to that of immunostaining with anti-β-protein. The new method also stained neurofibrillary tangles. Therefore, the methenamine-Bodian method could be widely used for the detection of senile changes in paraffin embedded sections from autopsied human brains.     

Effect of temperature on argyrophil impregnation: development of a high temperature rapid argyrophil procedure

Our studies on the effects of temperature on the demonstration of neurosecretory granules using argyrophil stains indicate an inverse relationship between the time needed for staining and temperature of the silver and reducing solutions. Careful monitoring of the temperature of silver solutions during the Grimelius procedure and its modifications show long incubation times serve in large part only to bring the solutions to reaction temperature. Tissue sections added when this temperature has been reached will stain with the same intensity as sections impregnated for the entire incubation period. We have modified the argyrophil procedure so that double-impregnation with solutions preheated to 60-70 C and development in Bodian's reducer prepared with preheated water rapidly demonstrates secretory granules. Our method does not require a microwave oven and much shorter incubation periods are required than with usual procedures. It is not necessary to incubate sections in hot solutions for extended periods of time, which can lead to detachment of sections, nonspecific staining and decomposition of the silver solution. Rinsing after impregnation and before development greatly increases contrast of argyrophil cells by reducing background staining. Our procedure results in more reliable staining of argyrophil and argentaffin cells and takes only ten minutes.     

Identification and Initial Characterization of Adipokinetic Hormone-Like Immunoreactive Peptides of Rat Origin

An antiserum was raised to adipokinetic hormone (AKH), a 10-amino-acid-residue peptide found in the arthropod Locusta migratoria. The antiserum demonstrated not only immunocytochemical reaction with some other arthropod species, but also stained many areas of the rat CNS, certain islet cells of the pancreas, and some anterior pituitary cells. The pattern of staining was unlike that for any known rat neuropeptide or hormone. With the antiserum used as the detection system, HPLC and high-voltage electrophoresis yielded two peptides that were purified to homogeneity from rat hypothalamic median eminence. These peptides have unique amino acid compositions, indicating they may be heretofore unknown rat neuropeptides.     

Differential Staining of Inner Ear Fluids by Protargol

Present day techniques for processing temporal bones involve celloidin embedding. With a few modifications in Bodian's silver staining procedure the celloidin of the endolymphatic spaces stains darker than that of the perilymphatic spaces providing there is no break in the anatomical barrier between them. Essentially the routine procedure of Bodian is used except that metallic copper is omitted from the staining solution, impregnation time is reduced to 3 hr, reduction time is extended to 10 min and no oxalic acid is used for gold toning.     

Immunological characterization of an anti-actin antibody specific for cytoplasmic actins and its use for the immunocytological localization of actin in Aplysia nervous tissue

Previous immunochemical and immunocytochemical studies have shown that an antibody to actin prepared from body wall muscle of the marine mollusc Aplysia californica is specific for vertebrate cytoplasmic actins. The ability of this anti-actin to distinguish between different forms of actin most likely reflects the recognition of amino acid sequences unique to cytoplasmic actins. We have confirmed the specificity of this antibody for cytoplasmic actins using nervous tissue as a source of cytoplasmic actin in further immunochemical studies. In addition to binding cytoplasmic actin in purified preparations, the antibody removed actin selectively from crude extracts of nervous tissue of some but not all of the species tested. Our results also suggest that tissue-specific differences in the distribution of cytoplasmic actins may exist. Immunofluorescence studies of Aplysia nervous tissue stained with anti-actin revealed that actin is present in the cell body and axonal processes of Aplysia neurons. Although the function of actin in nerve cells is not understood, the observed pattern of immunofluorescence staining is consistent with the idea that actin may be involved in movement within the axoplasm.     

Argentaffin cells in human adenohypophysis]

Appropriate techniques such as Bodian's method demonstrate numerous argyrophilic cells in the adenohypophysis of man. These cells were shown by subsequent staining methods to be chromophobe cells. Furthermore, beta cells may also be impregnated by the method of Bodian, but display a much less intense argyrophilia than the first mentioned cell type. Both cellular forms react positively to diazotation techniques used to demonstrate indol radicals. This findings should be related, for instance, with the demonstration of indolamines in beta cells by the technique of induced fluorescence.     

Panning and identification of a colon tumor binding peptide from a phage display peptide library

Tumor-targeting therapy can be an efficacious way to cure a malignant tumor in clinical trials. Phage display is a molecular diversity technology that allows the presentation of a large number of peptides or proteins on the surface of filamentous phage for various applications. In this study, we report on using phage display to generate peptide libraries that bind to colon cancer tissues. To accomplish this, we developed a screening protocol that contained 3 rounds of in vitro positive panning on colon cancer cells (SW480) and 2 rounds of subtractive screening in vitro on normal human intestinal epithelial cells with a phage display-7 peptide library. After several rounds of panning, both phage titer and recovery efficiency were significantly improved. Through a cell-based enzyme-linked immunosorbent assay, immunofluorescence, in vivo binding assay, immunocytochemical staining, and immunohistochemical staining, peptide CP15 (VHLGYAT) was demonstrated to be the most effective peptide in targeting tumor cells (SW480 and HT29 cells) and tumor tissues but not the normal human intestinal epithelial cells and control colon tissue. These studies suggest that peptide CP15 may be a promising lead candidate in the development of a useful colon tumor diagnostic and targeted drug delivery agent.     

Identification of cell types in Sertoli cell-enriched cultures by immunocytochemistry and DNA-specific fluorochrome Hoechst 33342

The cell types in Sertoli cell-enriched cultures can be identified by using the DNA-specific fluorochrome Hoechst 33342 staining. This simple, rapid and reproducible procedure can be used with fixed and living cells. The peritubular myoid cells can be distinguished from the Sertoli cells in Sertoli cell-enriched cultures by the characteristic staining pattern obtained using Hoechst 33342 dye. Those cells identified as peritubular myoid cells by the characteristic DNA staining also interacted with the anti-fibronectin antibody determined by an immunocytochemical method while the Sertoli cells did not. The described staining method is valuable in assessing the presence of peritubular myoid cells in Sertoli cell-enriched cultures.     

Increased sensitivity in immunocytochemistry. Effects of double application of antibodies and of silver intensification on immunogold and peroxidase-antiperoxidase staining techniques

Sensitivity and detection efficiency of immunocytochemical methods were tested on cytochemical models and tissue material, respectively. Use of silver intensification procedures revealed that staining with immunogold reagents could be rendered equally or even more sensitive than the standard peroxidase-antiperoxidase (PAP) method. Further increases in sensitivity with both methods could be obtained by double application of the primary antiserum. Combined use of the immunogold techniques and the PAP method with development in diaminobenzidine and subsequent silver intensification resulted in the most sensitive procedures. The procedures were applied to a wide variety of tissue preparations, including whole mount preparations of the external longitudinal muscle layer of the gut wall and were found not to produce any unspecific staining in any tissue tested. Use of immunogold-silver and, particularly of the combined immunogold-silver-PAP methods may be valuable for analyzing tissues and tumours containing small amounts of antigen, for testing the quality of immunogold staining procedures intended for ultrastructural studies and for electroblotting techniques.     

Chromogranin A: Immunocytochemical localization in secretory granules of frog atrial cardiomyocytes

Chromogranin A (CgA) belongs to the granin family of acidic proteins that are present in the secretory granules of many endocrine, neuroendocrine, and nerve cells. CgA has been shown to be stored in cardiomyocyte secretory granules of the rat heart atrium together with atrial natriuretic peptide (ANP). CgA-derived peptides (vasostatins) are known to produce a cardiosuppressive effect on isolated and working in vitro frog and rat hearts. Recently, CgA-derived vasostatin-containing peptides have been identified in rat hearts, whereas no data are available so far about the presence of CgA in the frog heart. In our work, we have studied the subcellular CgA localization in atrial myocytes of the adult frog R. temporaria heart by using an ultraimmunocytochemical method. Immunocytochemical staining of the frog atrial tissue for CgA and ANP showed the presence of the CgA-immunoreactive material in two types (A and B) of large specific atrial secretory granules, whereas no gold particles were revealed over the small granules (D) with a high electron density core. Similar results were obtained during the immunocytochemical staining by an antibody to ANP of the drog atrial cardiomyocytes. The data of the present work allow for the suggestion that CgA revealed in frog atrial cardiomyocytes, like CgA in rat cardiomyocytes, can be considered to be a precursor of intracardial vasostatins that, together with ANP, can play an important cardioprotector role under conditions of stress.     

Identification of cell types in sertoli cell-enriched cultures by immunocytochemistry and DNA-specific fluorochrome Hoechst 33342

Summary The cell types in Sertoli cell-enriched cultures can be identified by using the DNA-specific fluorochrome Hoechst 33342 staining. This simple, rapid and reproducible procedure can be used with fixed and living cells. The peritubular myoid cells can be distinguished from the Sertoli cells in Sertoli cell-enriched cultures by the characteristic staining pattern obtained using Hoechst 33342 dye. Those cells identified as peritubular myoid cells by the characteristic DNA staining also interacted with the anti-fibronectin antibody determined by an immunocytochemical method while the Sertoli cells did not. The described staining method is valuable in assessing the presence of peritubular myoid cells in Sertoli cell-enriched cultures.     

Co-expression of vimentin and cytokeratins in M cells of rabbit intestinal lymphoid follicle-associated epithelium

Summary Membranous epithelial (M) cells within the follicle-associated epithelium which overlies gut-associated lymphoid tissue in Peyer's patches and of appendix have been shown by immunocytochemical staining, in rabbit, to contain both vimentin- and cytokeratin-type intermediate filaments. The specificity of vimentin immunostaining has been confirmed by blocking with purified vimentin and by immunoblotting. No evidence was obtained for the expression of vimentin in rat, mouse or human M cells. The possible significance of vimentin-expression in these specialized epithelial cells and the potential use of vimentin as a positive marker for M cells are discussed.     

Immunohistochemical identification of Purkinje fibers and transitional cells in a terminal portion of the impulse-conducting system of porcine heart

Summary The ultrastructure of porcine ventricular tissue was studied by electron microscopy and immunocytochemical techniques. Electron-dense specific granules were found in both Purkinje fibers and transitional cells in the ventricular walls, and were positively stained by the immunogold staining method using an antiserum against atrial natriuretic polypeptide (ANP). This suggests that both the Purkinje fibers and transitional cells display the same specific granules as atrial cardiocytes containing ANP. These results demonstrate that Purkinje fibers and two types of transitional cells, in addition to the ordinary ventricular cardiocytes, can be identified in porcine ventricular wall tissue.