HBsAg, HBcAg and delta-Ag in liver tissue: simultaneous visualization in a single tissue section by triple immunostaining

The distribution pattern of HBsAg, HBcAg and Delta-Ag was investigated by immunohistochemistry in a series of paraffin embedded liver tissue specimens from 45 subjects with serum HBsAg and anti-Delta antibody positivity. An indirect immunoperoxidase technique was used. Stains for HBsAg, HBcAg and Delta-Ag were alternatively carried out on serial tissue sections and, for the first time, consecutively in a single section (triple immunostaining). Simultaneous presence of all antigens occurred in 7 out of 45 cases, and of two antigens (HBsAg and Delta-Ag) in the remaining 38. Two antigens (either HBsAg and HBcAg or HBcAg and Delta-Ag) could also be shown in the same cell. A series of new observations was forwarded by the successful application of triple immunostaining in the present series: 1) high frequence of simultaneous presence of HBcAg and Delta-Ag (7 out of 45 cases = 16%); 2) cytoplasmic localization of Delta-Ag; 3) localization of HBcAg on the cell membrane of HBsAg positive Ground-Glass (G-G) hepatocytes; 4) Ground-Glass appearance of hepatocytic cytoplasm associated with exclusive content of HBcAg (HBcAg-Ground-Glass: a new variant of G-G-hepatocytes).     

A novel technology allowing immunohistochemical staining of a tissue section with 50 different antibodies in a single experiment.

Immunohistochemical (IHC) examination is frequently necessary for a histological differential diagnosis of tumors. To simplify IHC examination, we have developed a novel device called a "multiplex-immunostain chip (MI chip)." The chip is a panel of antibodies contained in a silicon rubber plate that consists of 50 2-mm-diameter wells. A tissue section slide is placed on the plate and is fastened tightly with a specially designed clamp. The plate with the slide is then turned upside down, which applies the antibodies to the section. This technology allows IHC staining of a tissue section with 50 different antibodies in a single experiment, reducing the time, effort, and expense of IHC analysis. In addition, it enables pathologists to compare expression of multiple antigens on a tissue section simply by changing microscopic fields on a single slide. These features are unique to the MI chip technology. The method requires no expensive instruments. This device can be used in various applications in differential diagnosis of tumors and the field of cell biology.     

Simultaneous visualization by light microscopy of two pituitary hormones in a single tissue section using a combination of indirect immunohistochemical methods.

Two indirect methods involving enzyme-labeled antibodies were used to demonstrate simultaneously two distinct tissue antigens in the same histologic section without a need for antigen-antibody dissociative procedures. Sections of rat pituitary gland were incubated with rabbit anti-rat luteinizing hormone followed by goat anti-rabbit gamma-globulin conjugated to horseradish peroxidase. The same sections were then further incubated with monkey anti-rat growth hormone followed by goat anti-monkey gamma-globulin conjugated to glucose oxidase. Antigenic luteinizing hormone was subsequently localized with hydrogen peroxide-3,3'-diaminobenzidine as substrate for peroxidase, and growth hormone was localized with a glucose-phenazine methosulfate-nitroblue tetrazolium mixture as a substrate for glucose oxidase. The method relies on the availability of specific primary antibodies raised in different animal species in addition to corresponding specific secondary antibodies linked covalently to separate enzymes.     

A differential staining technique for simultaneous visualization of mitotic spindle and chromosomes in mammalian cells

A new technique has been devised for staining the mitotic spindle in mammalian cells while preserving spindle structure and chromosome number. The cells are trypsinized and fixed with a 3:1 methanol:acetic acid solution containing 4 mM MgCl2 and 1.5 mM CaCl2 at room temperature. The cells are then placed on slides and treated with 5% perchloric acid before staining with a 10% acetic acid solution containing safranin O and brilliant blue R. The preserved spindles appear dark blue against a light cytoplasmic background with chromosomes stained bright red. Individual chromosomes and chromatids are clearly visible. Positioning of the chromosomes relative to the spindle apparatus is readily ascertained allowing easy study of mitotic spindle and chromosome behavior.     

Principal cell types in the pancreatic islet of a teleost fish,Xiphophorus helleri H.

Summary By means of correlative light and electron microscopy, five pancreatic islet cell categories are described in the teleost fish, Xiphophorus helleri, each of which has specific light microscopic appearance and fine structure. Different histochemical techniques have been used, including immunofluorescence with antiporcine insulin and glucagon sera. In addition to B- and A₁-cells, two categories of A₂-cells have been observed, both reacting with antiporcine glucagon serum: A₂-cells with round granules gave a positive reaction for tryptophan; A₂-cells with crystalline granules gave a negative reaction with the same staining technique on the same section. The clear cells, the last category, were not specifically stained by any of the staining methods carried out in this investigation. The influence of fixation on staining affinities and on ultrastructure was shown to be considerable.Supported in part by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg (grant La 229/4) and by the Deutscher Akademischer Austauschdienst, Bonn-Bad Godesberg.     

The visualization of human erythrocyte membrane proteins and glycoproteins in SDS polyacrylamide gels employing a single staining procedure.

Human erythrocyte membrane proteins and glycoproteins were visualized after separation by sodium dodecyl sulfate polyacrylamide gels into molecular weight classes using a single staining procedure with a cationic carbocyanine dye (“Stains-all”). The sialoglycoproteins stained blue; the proteins, red; and the lipids, yellow-orange. This method is useful in detecting simultaneously the position of proteins and sialoglycoproteins in the commonly used SDS polyacrylamide gel electrophoresis.     

New types of islet cells in a cyclostome,Petromyzon marinus L.

Four types of acidophilic granular cells, in addition to B-cells, are identified in the islet organ of anadromous specimens of two subspecies of Petromyzon marinus by light and electron microscopy. Three of these acidophils (PI, PII and PIV-cells) occur in both the cranial and hepatic islets while a fourth type (PIII-cell) has only been found in the hepatic islet of some animals. The granules of the PI-cells stain with ponceau de xylidine, give a distinct tryptophan reaction and in ultrastructural examination show large, dense granules. The PII-cells contain unusual crystals and appear to be a non-secretory stage of the PI. The PIII-cells stain deep-red and acid fuchsin. They contain very large, dense granules and some lysosomes. PIV-cells stain selectively with phosphotungstic acid-hematoxylin and ultrastructurally, contain small, more or less dense granules. It appears that PI- and PIV-cells develop directly from B-cells, while the PIII-cells derive from PI-cells. despite their direct or indirect origin from B-cells, the PI-, PIII- and PIV-cells show characteristic features of functionally independent endocrine cells. Petromyzon marinus may be an ideal model for the understanding of phylogenetic and pathological interrelationships between islet and gastrointestinal hormones. It is clear that the interpretation of the islet organ of the cyclostomes, which has been generally considered a source of insulin only, requires a revaluation.     

Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining

Background  

Single-cell level studies are being used increasingly to measure cell properties not directly observable in a cell population. High-performance data acquisition systems for such studies have, by necessity, developed in synchrony. However, improvements in sample purification techniques are also required to reveal new phenomena. Here we assessed a cell sorter as a sample-pretreatment tool for a single-cell level assay. A cell sorter is routinely used for selecting one type of cells from a heterogeneous mixture of cells using specific fluorescence labels. In this case, we wanted to select cells of exactly the same size, shape, and cell-cycle stage from a population, without using a specific fluorescence label.     

Hematoporphyrin/DAPI staining: simplified simultaneous one-step staining of DNA and cell protein and trial application in automated cytological screening by flow cytometry

We describe a procedure for simplified, simultaneous one-step staining in 10 min for DNA and cell and tissue proteins using a newly developed staining solution containing 0.03% hematoporphyrin (HP) with 0.001% DAPI [or with Hoeschst 33342 (HO)]. These HP/DAPI or HP/HO solutions were especially developed to facilitate a trial of automated cancer cell screening on sputum samples using flow cytometry. Under UV light (365 nm) with fluorescence microscopy, HP/DAPI-stained cells showed red fluorescence (max. 670 nm) of cytoplasm and simultaneous blue fluorescence (max. 470 nm) of nuclei. The distance between the maximum peak of fluorescence spectra of DNA and that of protein was as large as 200 nm, and there was no detectable overlapping of each spectrum at the photometric filter range, which provided accurate measurement of DNA and protein. On flow cytometry, a single UV beam (370 nm) from the argon laser was used for excitation of both dyes. Measurement of DNA was done using a 470-nm bandpass filter and of protein using a 640-nm longpass (or 670-nm bandpass) filter. Reflecting the undetectable overlapping of the fluorescence spectra of protein and DNA, normal diploid cells in sputum revealed horizontal distributions along the 2C level on the dot-plot display of flow cytometry, which made sorting of abnormal hyperdiploid cells and cancer cells easier.     

Improved staining method for the simultaneous flow cytofluorometric analysis of DNA content, S-phase fraction, and surface phenotype using single laser instrumentation.

We have developed an improved technique for triple staining that permits the simultaneous flow cytofluorometric analysis of cell surface antigens, bromodeoxyuridine incorporation into DNA, and DNA quantification using 7-amino-actinomycin D. PHA-activated human peripheral blood lymphocytes were incubated with bromodeoxyuridine and stained for cell surface phenotype with phycoerythrin-labeled monoclonal antibodies. Stained cells were fixed serially with 1% paraformaldehyde and 45% ethanol. Fixed cells were sequentially stained with an anti-BrdUrd monoclonal antibody followed by a FITC-conjugated goat anti-mouse antibody and incubated with 7-amino-actinomycin D. Hypotonic buffer was employed for all procedures after fixation. Stained-fixed cells were analyzed by flow cytofluorometry for simultaneous green (525 nm), orange (570 nm), and red (greater than 650 nm) fluorescence. Utilizing this staining technique, we were able to analyze simultaneously cell phenotype, DNA synthesis, and total cellular DNA content with single laser excitation.     

Amyloid from a histochemical perspective. A review of the structure,properties and types of amyloid,and a proposed staining mechanism for Congo red staining

Amyloid is a diverse group of unrelated peptides or proteins that have positive functionality or are associated with various pathologies. Despite vast differences, all amyloids share several features that together uniquely define the group. 1) All amyloids possess a characteristic cross-ß pattern with X-ray diffraction typical of ß-sheet secondary protein structures. 2) All amyloids are birefringent and dichroic under polarizing microscopy after staining with Congo red, which indicates a crystalline-like (ordered) structure. 3) All amyloids cause a spectral shift in the peak wavelength of Congo red with conventional light microscopy due to perturbation of π electrons of the dye. 4) All amyloids show heightened intensity of fluorescence with Congo red, which suggests an unusual degree of packing of the dye onto the substrate. The ß portion of amyloid molecules, the only logical substrate for specific Congo red staining under histochemical conditions, consists of a stack of ß-sheets laminated by hydrophilic and hydrophobic interactions between adjacent pairs. Only the first and last ß-sheets are accessible to dyes. Each sheet is composed of numerous identical peptides running across the width of the sheet and arranged in parallel with side chains in register over the length of the fibril. Two sets of grooves are bordered by side chains. X grooves run perpendicular to the long axis of the fibril; these grooves are short (the width of the sheet) and number in the hundreds or thousands. Y grooves are parallel with the long axis. Each groove runs the entire length of the fibril, but there are very few of them. While Congo red is capable of ionic bonding with proteins via two sulfonic acid groups, physical constraints on the staining solution preclude ionic interactions. Hydrogen bonding between dye amine groups and peptide carbonyls is the most likely primary bonding mechanism, because all ß-sheets possess backbone carbonyls. Various amino acid residues may form secondary bonds to the dye via any of three van der Waals forces. It is possible that Congo red binds within the Y grooves, but that would not produce the characteristic staining features that are the diagnostic hallmarks of amyloid. Binding in the X grooves would produce a tightly packed series of dye molecules over the entire length of the fibril. This would account for the signature staining of amyloid by Congo red: dichroic birefringence, enhanced intensity of fluorescence and a shift in visible absorption wavelength.     

Inhibition reactivation myofibrillar ATPase technique for demonstration of three fiber types in a single cryostat muscle section

An inhibition reactivation technique was used for histochemical staining of human skeletal muscle sections. Myofibrillar ATPase activity was inhibited by sodium hydroxymercuribenzoate (2.5 mM in 0.1 M Tris-HCl buffer, pH 7.2-7.5, 30 min) and successively reactivated by cysteine which was added to incubation solution (10 mM cysteine-HCl, 2.5 mM ATP-disodium salt, 50 mM potassium chloride and 27 mM calcium chloride in barbital buffer, pH 9.4, 35 min at 37 C). This technique allows the distinction of three fiber categories with different staining intensities in single cross-section. Dark, intermediate and light fibers correspond to IIB, I, and IIA types, respectively. Storage of air dried sections in the freezer at -20 C for one month had no influence on staining characteristics.     

A successive histochemical staining for succinate dehydrogenase and "reversed"-ATPase in a single section for the skeletal muscle fibre typing

A procedure is described which simplifies the classification of skeletal muscle fibres in that it allows a simultaneous evaluation of both the oxidative capacity and the intensity of "reversed" ATPase of the fibres, and thus enables to distinguish three fibre types - SO, FOG and FG - in one tissue section. After preincubation at pH 4.1-4.2 the cryostat section is incubated for succinate dehydrogenase (SDH) and subsequently for "reversed"-ATPase. This is followed by the fixation with neutral buffered formaldehyde. The results of typing of chicken, minipig and rabbit fibres in a single muscle section stained with this technique are identical to those obtained with the usual method based on a comparison of serial sections of which one is stained for SDH activity the other for "reversed"-ATPase activity.     

Osmium ferricyanide fixation improves microfilament preservation and membrane visualization in a variety of animal cell types

Using a fixation formula which includes adding potassium ferricyanide (K3Fe(CN)6) to the osmium step and an en bloc aqueous uranyl acetate step before dehydration we have looked at cells from mammals, birds, amphibia, algae, and higher plants and we have collaborated in fixing cells of teleost fish. In every cell type except the algae and higher plants the final EM image was improved by the OsFeCN-uranium method. The most common improvement was an increase in the membrane contrast but more significantly, some cells show improved preservation of microfilaments. We conclude that the OsFeCN adds contrast to all classes of membrane and does not destroy microfilaments to the extent that osmium alone does. Adding uranyl acetate to the cells may protect delicate filamentous structures from collapse during dehydration and embedding. We have preliminary evidence in PtK1 cells that addition of tannic acid after OsFeCN may function in a similar manner. This method is recommended for any animal cell type where improved visualization of membranes and filaments is required.     

Identification of cell types containing S-100b protein-like immunoreactivity in the islets of Langerhans of the guinea pig pancreas with light and electron microscopy

Summary Cell types containing S-100b protein-like immunoreactivity in the islets of Langerhans of the guinea pig were studied by light- and electron-microscopic immunocytochemistry using antisera to S-100b protein, insulin, glicentin, somatostatin, and pancreatic polypeptide. Two types of S-100b-immunoreactive cells were identified. The first type was stellate and characterized by thin cytoplasmic processes sheathing endocrine-type cells, especially pancreatic A-cells. It was located predominantly in the neuro-insular complex and in large islets, both of which were located near the main pancreatic duct. Intense immunoreactivity was found in the cytoplasmic matrix as well as in the nucleoplasm. Nerve fibers or endings were occasionally ensheathed by its cytoplasmic processes. The second type, whose immunoreactivity was rather weak and varied from one cell to another, was oval to polygonal in shape and located randomly throughout the islets. It was an endocrine cell-type and its immunoreactivity was located in the secretory granule. With the use of immunostained consecutive sections for demonstrating pancreatic endocrine cell-types, it was found that a portion of the pancreatic B-cell population expressed S-100b-like immunoreactivity.     

Activation and expression of ERK, JNK, and p38 MAP-kinases in isolated islets of Langerhans: implications for cultured islet survival.

Isolation and purification of islet cells exposes them to ischemic, osmotic and mechanical stresses. The objective of this study was to determine the roles of the MAP-kinases in islets immediately following isolation. During the first 48 h, activity of JNK1 and JNK2 declined markedly. Activity of p38 increased steadily with time in culture while extracellular signal regulated kinase (ERK) activity declined dramatically within 24 h post-isolation. High p38 activation relative to ERK activation immediately following isolation correlated with a decrease in islet survival after 36 h in culture. Absence and/or transiency of ERK signaling in conjunction with sustained activation of p38 pathway could be an important regulator of cell death in islets during and following their isolation by commonly employed procedures.