Simultaneous demonstration of multiple antigens by indirect immunofluorescence or immunogold staining

Summary Available techniques for light and electron microscopical double immunocytochemical staining are all associated with certain problems. We have developed a novel multiple staining procedure, which allows use of antibodies of differing specificities, raised in the same species (e.g. rabbit). Its essential features include 1) saturation of antigenic epitopes on the first layer primary antiserum by second (fluorophor- or gold-) labelled anti-IgG antibodies and 2) denaturation of free anti-IgG binding sites by formaldehyde vapour treatment. Various combinations of gastrin, somatostatin, glucagon, ACTH, growth hormone and enkephalin/endorphin antibodies have been tested at the light and electron microscopical level and have been found to give highly reproducible double- and triple-staining results. The technique has also been evaluated by use of cytochemical paper models. The method is simple and very useful for multiple staining of a wide variety of antigens.     

Microwave applications in classical staining methods in formalin-fixed human brain tissue: a comparison between heating with microwave and conventional ovens.

The quality of microwave adaptations of three classical neuroanatomical staining methods (the Nissl, Klüver-Barrera and H?ggqvist stains) was tested on frozen serial sections from human brain specimens which has been stored for up to 10 years in 10% formalin. The conclusion was that the use of microwave irradiation reduces processing time and/or concentrations of the chemicals used, whereas the light microscopical quality of the stains considered is equal or improved as compared to their original counterparts. Next, a comparison was made between microwave adapted stains and classical procedures, which, except for the use of a conventional oven as heat source together with pre-heated solutions, were entirely identical. It appeared, that at light microscopical level no difference can be appreciated between the effect of internally (using microwave irradiation) and externally (using a conventional oven) supplied heat on the staining result.     

Negative staining in the detection of viruses in clinical specimens

Viruses have unique morphology and are therefore good candidates for negative staining. Negative staining with phosphotungstic acid (PTA) or uranyl acetate has facilitated the detection of many viruses in clinical specimens. Enhancement procedures have included the use of centrifugation and agar diffusion for concentrating virus particles, the use of solid phase capture reagents to trap virus particles and the use of secondary antibodies and electron dense markers to help visualize them. Techniques currently in use and employing negative staining include direct EM, immune electron microscopy (IEM), solid phase immune electron microscopy (SPIEM), colloidal gold-labeled protein A (PAG), solid phase IEM employing a second decorator antibody (SPIEMDAT), and solid phase IEM using colloided gold-labeled secondary antibodies (SPEIMDAGT). IEM methods assist with the detection of small viruses or viruses present in low numbers while PAG offers increased sensitivity over direct EM and IEM. In our experience the serum-in-agar (SIA) method is the most sensitive of the PAG IEM techniques for detection of rotavirus particles in clinical specimens. SPIEMDAT enhances the detection of small viruses which are often missed by other techniques due to background staining in specimens. SPEIMDAGT employing colloidal gold-labeled secondary antibody has increased sensitivity and offers the advantage of detecting viral antigen when whole virus particles are not visible. IEM techniques have recently been used for typing viruses using either monospecific antisera or monoclonal antibodies and colloidal gold-labeled secondary antibody.     

Combination of the peroxidase anti-peroxidase (PAP)- and avidin-biotin-peroxidase complex (ABC)-techniques: an amplification alternative in immunocytochemical staining.

A combination of the PAP- and ABC-techniques was developed to enhance the intensity of the immunocytochemical staining with monoclonal antibodies at light and electron microscopical levels. This amplification technique could be performed in 4 (single PAP + ABC) or 6 (double PAP + ABC) sequential steps depending on the quality of the primary antibodies used and the processing of the tissue before the immunocytochemical reaction: First step--Incubation of the tissue sections with the monoclonal primary antibodies; Second step--biotinylated anti-rat or anti-mouse IgG; Third step--monoclonal PAP complex; Fourth step--ABC complex which binds to the biotinylated secondary antibody. If stronger enhancement of the immunostaining has required the steps 2 and 3 could be repeated followed by the 6th step--the ABC complex. Choline acetyltransferase-like immunoreactivity of the rat hypoglossal nucleus and desmin- and vimentin-like immunoreactivity of human testis were studied. After the 4- and more pronounced the 6-step reaction a significant increase of the staining intensity was observed for all the reactions under study. ChAT-like immunoreactivity was observed to longer distances of the nerve cell dendrites after their emerging from the perikarya and within a greater number of structures in the neuropil as compared to the standard techniques. At electron microscopical level the technique permits longer fixation of the tissue which is important for the better preservation of the ultrastructure as well as for the easier recognition of the reaction product even in the smallest dendrite branches and the axons of the nerve cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Light microscopical detection of antigens and lectin binding sites with gold-labelled reagents on semi-thin Lowicryl K4M sections: Usefulness of the photochemical silver reaction for signal amplification

Summary In the present study, we have investigated the applicability of semi-thin sections from low temperature Lowicryl K4M-embedded tissues for cytochemical labelling with protein A—gold and lectin—gold complexes. In order to ensure the best possible signal-to-noise ratio antibodies, protein A—gold and lectin—gold were applied in concentrations used for labelling at the electron microscope level. Furthermore, due to the lack of an appropriate chemical procedure for resin removal, untreated semi-thin sections were incubated. Under such conditions, semi-thin sections displayed either no visible staining or only a faint incomplete staining. However, following photochemical silver reaction, the latent or faint incomplete staining was rendered visible in most cases. It is concluded that the same block of Lowicryl K4M-embedded tissue and the same labelling reagents can be used for both light and electron microscopical cytochemical studies. At the light microscopical level, a high degree of structural and specific staining information is obtained. The reactivity of cellular components with antibodies or lectins is preserved even after years of storage of the blocks or slides containing semi-thin sections.     

Combination of the peroxidase anti-peroxidase (PAP)-and avidin-biotin-peroxidase complex (ABC)-techniques: an amplification alternative in immunocytochemical staining

Summary A combination of the PAP- and ABC-techniques was developed to enhance the intensity of the immunocytochemical staining with monoclonal antibodies at light and electron microscopical levels. This amplification technique could be performed in 4 (single PAP + ABC) or 6 (double PAP + ABC) sequential steps depending on the quality of the primary antibodies used and the processing of the tissue before the immunocytochemical reaction: First step — Incubation of the tissue sections with the monoclonal primary antibodies; Second step — biotinylated anti-rat or anti-mouse IgG; Third step — monoclonal PAP complex; Fourth step — ABC complex which binds to the biotinylated secondary antibody. If stronger enhancement of the immunostaining has required the steps 2 and 3 could be repeated followed by the 6th step — the ABC complex. Choline acetyltransferase-like immunoreactivity of the rat hypoglossal nucleus and desmin- and vimentin-like immunoreactivity of human testis were studied. After the 4-and more pronounced the 6-step reaction a significant increase of the staining intensity was observed for all the reactions under study. ChAT-like immunoreactivity was observed to longer distances of the nerve cell dendrites after their emerging from the perikarya and within a greater number of structures in the neuropil as compared to the standard techniques. At electron microscopical level the technique permits longer fixation of the tissue which is important for the better preservation of the ultrastructure as well as for the easier recognition of the reaction product even in the smallest dendrite branches and the axons of the nerve cells. Stronger staining intensity was obtained for desmin-like immunoreactivity (LI) (within myofibroblasts of the lamina propria and muscle cells of the blood vessels)-and vimentin-LI (within Sertoli cells, myofibroblasts of the lamina propria and fibroblasts of the interstitium) of the human testis. The full combination (6 step-reaction) permits the detection of smallest quantities of an antigen in sections of different tissues using highly diluted primary antibodies. The technique represents a further alternative among the immunocytochemical amplification methods.     

Reflection contrast microscopy of ultrathin sections in immunocytochemical localization studies: a versatile technique bridging electron microscopy with light microscopy

Reflection contrast microscopy (RCM) of ultrathin sections was recently introduced as a sensitive technique for visualization with enhanced definition in immunogold histochemistry. Experience of using RCM as a major tool in immunocytochemical research in different fields is summarized, e.g. oncology, nephrology and embryology. The sensitive visualization of immunocytochemical labels, gold particles or peroxidase-diaminobenzidine deposits in or on ultrathin sections, by RCM instead of electron microscopy is demonstrated. RCM of ultrathin sections is an adequate light microscopical alternative for immunoelectron microscopy, since an overview of both label and tissue is obtained with a high image definition and high contrast of label. In the studies presented, RCM is shown to provide a better gradation in staining intensity and staining pattern than other light microscopical methods. Moreover, a precise localization of multiple labels is obtained with this method. Besides the applications shown, ultrathin section visualization by RCM is very useful for correlative light- and electron microscopical studies of fine structures. Commercially available fluorescence microscopes can be adapted for proper RCM functioning; an adaptation scheme and list of microscopes tested is provided.     

Differential staining of neutrophils and monocytes: Surface and cytoplasmic iron-binding proteins

Summary Lactoferrin, transferrin, and ferritin were systematically visualized and semiquantified in neutrophils and monocytes/macrophages using indirect immunofluorescence and functional cytochemical techniques. They localized on cell surfaces and within the cytoplasm at the light and electron microscopical levels. In normal subjects, subpopulations of blood neutrophils and monocytes had surface lactoferrin, but little surface transferrin or ferritin was observed on these cells. Most neutrophils had brilliant granular cytoplasmic positivity for lactoferrin; variable fractions of monocytes had weak to moderate diffuse cytoplasmic lactoferrin staining localized most prominently to the cytoplasmic matrix. Most neutrophils had cytoplasmic ferritin, but few had cytoplasmic transferrin, whereas larger subpopulations of monocytes had cytoplasmic staining reactions for both proteins. To analyse maturing cells, the iron nitrilotriacetate-acid ferrocyanide method was adapted for the light microscopical analaysis of neutrophils and monocytes/macrophages in soft agar culture. Further, a combined stain that visualizes iron nitrilotriacetate-acid ferrocyanide reactivity and -naphthyl butyrate esterase activity in cells in blood and marrow smears was developed. The relative quantities and subcellular distribution of iron-binding proteins in neutrophils and monocytes/macrophages defined by the present methods can be correlated with biochemical, maturational, and functional properties of these cells.     

Purification of mouse alpha1-fetoprotein and preparation of specific peroxidase conjugates for its cellular localization.

Mouse alpha1-fetoprotein (AFP) was isolated from amniotic fluid by immunoadsorbent columns and preparative electrophoresis. Specific antibodies were isolated from monospecific hyperimmunsera by use of immunoadsorbents, and subsequently coupled with horseradish peroxidase. At the light microscopical level, purified antibody-peroxidase conjugates were used for the cellular localization of AFP in fetal liver by direct and indirect staining methods. Fixatives containing ethanol or aldehydes were tried for antigen staining. Prior to immunocytological reactions, endogenous peroxidases were inhibited by hydrogen peroxide.     

Enzyme markers: their linkage with proteins and use in immuno-histochemistry

Synopsis The preparation and use of enzyme-labelled antibodies and antigens is described. First, the enzyme is linked covalently to the antibody or antigen. Next, the enzyme-labelled protein is allowed to react with the cellular antigen or antibody; and finally, the sites of bound enzyme are revealed with appropriate cytochemical staining techniques at either the light or electron microscopical levels.Because the specific activity of an enzyme can be assayed by appropriate enzymological techniques, enzyme-labelled antibodies can also be employed for measuring the amounts of cellular constituents. Similarly, enzyme-labelled antigens can be used for the quantitation of humoral antigens.In addition to the antigen-antibody reaction, enzyme markers can also be used for the quantitation and localization of other specifically interacting constituents.     

Neuron specific enolase (NSE) immunostaining a useful tool for the light microscopical detection of endocrine cell hyperplasia in adult rats exposed to asbestos

Summary Hyperplasia of endocrine cells in the lung of the adult rat exposed to asbestos has only been characterised so far by electron microscopy as there is a lack of reliable staining techniques for their demonstration at light microscopical level. Neuron specific enolase (NSE), an isoenzyme of the glycolytic enzyme enolase has recently been shown to be present in lung endocrine cells. In this study we reveal a marked endocrine cell hyperplasia at light microscopical level in the lungs of adult rats exposed to asbestos using antibodies to NSE. Very large groups of NSE-immunoreactive cells (20–80) were only observed in the lungs of rats exposed to asbestos for 12 months. In addition smaller groups of cells (2–10) known to be present normally and to decrease with age, were rarely noted in the controls but were frequently detected in the treated rats. Immunoreactive NSE is therefore a very good marker for endocrine cell hyperplasia and thus of early neoplastic changes.     

Immunogold-silver staining method for light and electron microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies

We used the immunogold-silver staining method (IGSS) for detection of lymphocyte cell surface antigens with monoclonal antibodies in light and electron microscopy and compared this procedure with the immunogold staining method. Two different sizes of colloidal gold particles (5 nm and 15 nm) were used in this study. Immunolabeling on cell surfaces was visualized as fine granules only by IGSS in light microscopy. The labeling density (silver-gold complexes/cell) and diameters of silver-enhanced gold particles on cell surfaces were examined by electron microscopy. Labeling density was influenced not by the enhancement time of the physical developer but by the size of the gold particles. However, the development of shells of silver-enhanced gold particles correlated with the enhancement time of the physical developer rather than the size of the colloidal gold particles. Five-nm gold particles enhanced with the physical developer for 3 min were considered optimal for this IGSS method because of reduced background staining and high specific staining in the cell suspensions in sheep lymph. Moreover, this method may make it possible to show the ultrastructure of identical positive cells detected in 1-micron sections counterstained with toluidine blue by electron microscopy, in addition to the percentage of positive cells by light microscopy.     

Double immunogold staining method for the simultaneous ultrastructural localization of regulatory peptides

Recent studies have suggested that the morphological characteristics of secretory granules contained within endocrine cells and nerves may be determined largely by their chemical composition. The use of the immunogold staining (IGS) method, which is based on the adsorption of colloidal gold to immunoglobulins, has been used in our laboratory to demonstrate a wide range of intracellular antigens at both the light and electron microscope levels. In this study we have applied a modification of the IGS method for the simultaneous detection of two separate antigens in a single tissue section, using a variety of region-specific antisera to different peptides. Peptide antisera were raised in rabbits or in guinea pigs and these were applied simultaneously or sequentially to grid-mounted ultrathin tissue sections. Antigenic sites were visualized at the electron microscope level using antisera raised in goats, adsorbed to gold particles of 12, 20, or 40 nm. Using this technique we have attempted to investigate the coexistence of multiple antigens in single tissue sections, in particular in single granules; the topographic distribution of molecular forms within one single granule or granule population; the heterogeneity of peptidergic neurons and also the heterogeneity of peptide content in morphologically similar granules. The double immunogold staining procedures described here have proved to be extremely effective for the simultaneous ultrastructural localization of two antigens (peptide-peptide; peptide-propeptide) on a single tissue section. The further development of this technique may provide useful information on neuroendocrine cell dynamics in normal and diseased states.     

A fixation method for improved antibody penetration in electron microscopical immunoperoxidase studies.

A fixation method for electron microscopical immunoperoxidase staining has been developed, which (a) allows penetration of antibodies through cell membranes to intracellular antigen sites, (b) provides a reasonable cell preservation and (c) does not alter the antigenic structure in too great an extent. Penetration of the antibodies has been achieved by using saponin as a cell membrane attacking agent. The best results could be obtained after pretreatment of cell monolayers with a mixture of 0.05% saponin, 0.0125%-0.05% glutaraldehyde and 1% paraformaldehyde for 5 min at 4 degrees C, and postfixing them with the corresponding fixative without saponin for 45 min at 4 degrees C.     

Appearance of nuclear pore complexes after Bernhard's staining procedure

Summary Bernhard's electron microscopical differential staining procedure for nucleoproteins (1969) was employed with plant (onion root tip) and animal (HeLa) material for a detailed study of the nuclear pore complex structures. All non-membraneous components such as the annular and the inner pore material including the central granule and the pore-associated fibrils are strongly stained in contrast to the adjacent chromatin. No difference in staining behaviour was observed between the different components. In general, however, the pore complex structures showed slightly less contrast than the cytoplasmic ribosomes and the nucleolar particles with which they appear to be connected in fibrillar continuity. Although this preferential staining speaks against a considerable deoxyribonucleoprotein content of the structures in question, it cannot serve to differentiate between ribonucleoproteins and pure protein structures.The work was supported in part by the Deutsche Forschungsgemeinschaft.The authors are indebted to Miss Sigrid Krien for careful technical assistance as well as to Dr. Hans Kleinig for help with the HeLa material.     

Localization of somatostatin receptors at the light and electron microscopial level by using antibodies raised against fusion proteins

Somatostatin mediates its multiple biological effects via specific plasma membrane receptors belonging to the family of G-protein coupled receptors with seven putative membrane-spanning domains. Five somatostatin receptor subtypes (sst1-sst5) have been cloned in human, mouse, and rat. We have raised specific antibodies against the five human somatostatin receptors by using the fusion protein technique. DNA sequences encoding C-terminal parts of the somatostatin receptors were inserted into a pGEX-2T plasmid vector. E. coli bacteria were transformed with the recombinant plasmid and fusion proteins were expressed and purified using the glutathione S-transferase Gene Fusion System. The fusion proteins were emulsified with Freund's complete adjuvant and polyclonal antibodies were raised in rabbits. The antisera were tested for specificity in Western blot analysis of membrane preparations from cell lines expressing the receptors and in membrane preparations of brain tissues. The receptors were visualized at the light microscopical level in paraformaldehyde fixed tissue sections by use of biotin labelled secondary antibodies as well as by amplification with biotinylated tyramide. The final step in the immunohistochemical visualization of the receptors was done by both peroxidase labelled streptavidin/biotin and different fluorophores. At the electron microscopical level, some of the receptors could be visualized in tissues fixed with a combination of paraformaldehyde and low concentrations of glutaraldehyde. In the hamster brain, sst2 receptors labelling was observed in both neuronal processes and perikarya. The staining was present in neo-, and allocortical areas of the forebrain, the hypothalamus, brain stem, and spinal cord. In the rat and human, sst1 receptor was shown to be an auto receptor on somatostatinergic neurons located in the hypothalamus. In the retina both sst1 and sst2 receptors were present. sst1 receptors were confined to amacrine cells, few ganglionic cells, and Müller cell-end feet. sst2 receptors were more widespread than the sst1 receptors. sst2-immunoreactivity was present in dopaminergic amacrine cells, the Müller cell-end feet, and in the inner segments of the cone photoreceptors. Thus, the availability of subtype specific antibodies against the five somatostatin receptors makes it possible to identify the receptors involved in the multiple somatostatinergic system in the body.     

Basal lamina formation by porcine thyroid cells grown in collagen- and laminin-deficient medium

Summary Porcine thyroid cells isolated by dispase treatment were cultured in either (a) Matrigel, (b) agarose with the addition of different combinations of basic fibroblast growth factor and laminin, or (c) on agarose-coated dishes. The formation of follicles and the presence of a basal lamina was investigated by routine electron microscopy of Araldite-embedded material and by light and electron microscopical immunocytochemical detection of the basal lamina components, laminin and collagen type IV. After 10 days of culture in Matrigel or agarose, a basal lamina-like structure surrounded most follicles. Follicles of cells growing in agarose and overlaid with a medium containing thyrotropin and fibroblast growth factor showed a fluorescent band at the basal side of the follicles after immunocytochemical staining with anti-laminin and anti-collagen antibodies. Routine electron microscopy showed that a basal lamina-like structure lined the outside of the follicle. This structure could be subdivided into a lamina lucida and a lamina densa. Electron microscopical immunogold labelling revealed that immunologically detectable laminin was confined to the lamina densa. These findings suggest that even in the absence of basal lamina components in the culture medium, thyroid cells are able to form follicles with a regular basal lamina when they are cultured in a three-dimensional environment.     

Preparation of hybrid antibodies with mouse IgG and ferritin specifities for the immune electron microscopic detection of cell membrane antigens]

Use of hybrid antibodies with one specifity for mouse-IgG and the other one for ferritin is particularly suitable for immune electron microscopical detection of cell surface antigens. Preparation of antibodies of such kind is described, whereby the method introduced by HMMERLING et al. is varied within some steps of preparation. These variations are discussed here. Activity of produced hybrid antibodies is demonstrated by labeling the THY 1.1. antigen on the cell surface of the thymocytes of the mouse. The advantages of utilizing the hybrid antibodies in comparison with known immune electron microscopical techniques are an excellent location of the antigens, the possibility of using distinct particles for labeling, the application of a multiple labeling, and the fact that investigations by both the transmission and the scanning electron microscope can be carried out by means of the same preparation of hybrid antibodies.     

Peroxidase-labeled antibody staining for carcinoembryonic antigen of cytologic specimens for light and electron microscopy

Immunohistochemical staining methods suitable for light and electron microscopic examination of cytologic specimens are described. Application of the methods clearly demonstrated the localization of carcinoembryonic antigen (CEA) in adenocarcinoma cells in body fluids. The use of a peroxidase-labeled antibody method permits rapid penetration of the cells by the antibody, which is not achieved by the peroxidase-antiperoxidase or avidin-biotin-peroxidase-complex staining methods. Since mesothelial and inflammatory cells are negative for CEA, the staining of body fluids for CEA is expected to be an extremely useful tool for the differential diagnosis of adenocarcinoma.     

Criteria of reliability for light microscopic immunocytochemical staining

Summary The following criteria of reliability are defined and discussed for immunocytochemical staining at the light microscopical level: efficiency, accuracy, precision, sensitivity, and specificity. Whenever practical, tests are suggested for obtaining information on the extent to which these criteria are fulfilled in a given system, and procedures are outlined for improving immunocytochemical staining in terms of these criteria. It is suggested that consideration of reliability criteria will help investigators in their choice of methodology, design of experimental strategy, and valid interpretation of the results.