Ultrastructural immunocytochemistry of glia cells

Summary The introduction of acrylate resins (Lowicryl K 4M, LR White) into electronmicroscopic immunocytochemistry applied to embedded tissue (post-embedding method) has improved the localization of antigens because of a satisfactory preservation of both ultrastructure and antigenicity of tissues. Here we describe a method that allows double staining of intracellular and membranous determinants in ultrathin sections of nervous tissue and cultures of peripheral nervous system cells. Ultrathin sections of the rat central nervous system fixed on uncoated grids were stained first for MBP selectively on the one face, then the opposite face was stained for GFAP using monoclonal antibodies and indirect immunogold staining method (IGS). Cultured Schwann cells induced to express major histocompatibility complex (MHC) class II antigens were stained for class. H antigens by pre-embedding method then followed by post-embedding IGS for the other intracytopasmic antigens.The Clinical Research Unit for Multiple Sclerosis is supported by Hermann and Lilly Schilling foundation     

Preembedding colloidal gold immunostaining of hypothalamic neurons: light and electron microscopic localization of beta-endorphin-immunoreactive perikarya

A preembedding immunogold staining (IGS) procedure was developed to identify beta-endorphin/adrenocorticotropic hormone immunoreactive neurons at the light and electron microscopic levels. Colchicine-treated rats were perfused with Nakane's periodate-lysine-paraformaldehyde fixative. Vibratome sections were incubated in primary antisera followed by goat anti-rabbit immunoglobulin G coupled to 16 nm colloidal gold, and, in some cases, rabbit immunoglobulin G coupled to gold. The appearance to pink to light red perikarya, corresponding to colloidal gold deposition at antigenic sites, was monitored under the light microscope. Positive cell bodies in the arcuate region sometimes extended lateral to the nucleus. Only proximal portions of neuronal processes were stained. At the ultrastructural level, colloidal gold labeled the periphery of 90-110 nm dense neurosecretory granules in the perikaryal cytoplasm and a few proximal axons. Clusters of gold particles, appearing free in the neuroplasm, actually labeled secretory granules in adjacent thin sections. Granules associated with the Golgi apparatus were not stained. Colloidal gold labeling of mature beta-endorphin granules, but not progranules, in rat hypothalamic neurons was confirmed using the peroxidase-antiperoxidase technique. The results correlate well with data on the intracellular processing of pro-opiomelanocortin in pituitary cells and prepropressophysin in the paraventricular nucleus. These data demonstrate the first application of the preembedding colloidal gold staining method for the identification of intracellular antigens within the central nervous system. The IGS method provides a definitive marker for single or double labeling of nervous tissue at both the light and electron microscopic levels.     

Immuno gold staining (IGS) for electron microscopical demonstration of glial fibrillary acidic (GFA) protein in LR white embedded tissue

Post-embedding immunocytochemical staining methods using gold have so far failed to label intermediate filament antigens in situ in epon or araldite embedded tissue. We have now applied the post-embedding immuno gold staining (IGS) technique for LR White embedded tissue. Glial fibrillary acidic (GFA) protein immunoreactivity was clearly demonstrated electron microscopically on astrocytic filaments of rat cerebellum in situ.     

Immunogold electron microscopic localization of glial fibrillary acidic protein (GFAP) in neurohypophyseal pituicytes and tanycytes of the Mongolian gerbil (Meriones unguiculatus)

The post-embedding immuno gold staining (IGS) technique was used for the ultrastructural localization of glial fibrillary acidic protein (GFAP) in pituicytes and tanycytes of the neurohypophysis. IGS was applied to LR White embedded neurohypophyseal tissue of the Mongolian gerbil (Meriones unguiculatus), a species which contains abundant GFAP-positive pituicytes and tanycytes. GFAP-immunoreactivity could be demonstrated on pituicytic intermediate filaments (IF's) in situ. Thus, it was shown that pituicytes contain GFAP in its filamentous form, what had been a matter of speculation. At the ultrastructural level, gerbil tanycytes and tanycyte-like cells in the external zone of the median eminence were characterized by a great amount of densely packed IF's, which were labeled by both GFAP- or vimentin-antibodies. Sequential immunostaining of serial semithin sections with GFAP- and vimentin-antibodies revealed an invariable coexpression of the two IF proteins in somata and processes of these median eminence cells.     

Immunogold electron microscopic localization of glial fibrillary acidic protein (GFAP) in neurohypophyseal pituicytes and tanycytes of the Mongolian gerbil (Meriones unguiculatus)

Summary The post-embedding immuno gold staining (IGS) technique was used for the ultrastructural localization of glial fibrillary acidic protein (GFAP) in pituicytes and tanycytes of the neurohypophysis. IGS was applied to LR White embedded neurohypophyseal tissue of the Mongolian gerbil (Meriones unguiculatus), a species which contains abundant GFAP-positive pituicytes and tanycytes. GFAP-immunoreactivity could be demonstrated on pituicytic intermediate filaments (IF's) in situ. Thus, it was shown that pituicytes contain GFAP in its filamentous form, what had been a matter of speculation. At the ultrastructural level, gerbil tanycytes and tanycyte-like cells in the external zone of the median eminence were characterized by a great amount of densely packed IF's, which were labeled by both GFAP- or vimentin-antibodies. Sequential immunostaining of serial semithin sections with GFAP- and vimentin-antibodies revealed an invariable coexpression of the two IF proteins in somata and processes of these median eminence cells.     

Immuno gold staining (IGS) for electron microscopical demonstration of glial fibrillary acidic (GFA) protein in LR White embedded tissue

Summary Post-embedding immunocytochemical staining methods using gold have so far failed to label intermediate filament antigens in situ in epon or araldite embedded tisue. We have now applied the post-embedding immuno gold staining (IGS) technique for LR White embedded tissue. Glial fibrillary acidic (GFA) protein immunoreactivity was clearly demonstrated electron microscopically on astrocytie filaments of rat cerebellum in situ.Abbreviations BSA Bovine serum albumin - DAB 3,3-diaminobenzidine - GAM G10 Goat anti-mouse IgG gold particle size 10 nm - GFAP Glial fibrillary acidic (GFA) protein - IGS Immuno gold staining - PBS Phosphate buffered saline - TRITC Tetramethylrhodamine isothiocyanate     

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.     

Immunohistochemical detection of strain-specific major histocompatibility complex class I antigens in paraffin-embedded rat osteochondral tissue

Summary An immunohistochemical method using formalin-fixed, paraffin wax-embedded sections is described for detecting strain-specific major histocompatibility complex class I antigens in knee-joint tissue from DA and Lewis strains of rat. The fixed osteochondral tissues were additionally decalcified in formic acid before processing for paraffin wax embedding. For immunohistochemistry, two monoclonal antibodies, one specific for DA class I allele RT1Aa and the other for Lewis class I allele RT1A1, were used together with the avidin-biotin immunoperoxidase procedure. It was necessary to use strain-specific normal rat serum as a diluent for the antibodies to suppress cross-strain recognition. DA-specific antibody stained positively only on DA rat sections, not on Lewis rat sections, and Lewis-specific antibody stained positively only on Lewis rat sections, and not on DA. Positive staining was localized in the bone marrow, osteochondral cells and endothelium. We propose that the use of a decalcification medium may have enhanced the immunoreactivity of the tissue. The method described can be used on sections of allografts from the two strains of rat to assess morphologically the extent of cellular replacement of the graft by the host's cells.     

Post-embedding staining with high-iron diamine-thiocarbohydrazide-silver proteinate and its application to visualizing sulfated glycoconjugates in cryofixed kidney and cartilage.

Cryofixation is generally believed to provide optimal tissue preservation. However, certain post-embedding cytochemical reactions, such as high-iron diamine (HID) staining for sulfated glycoconjugates, are not applicable to cryofixed and freeze-substituted tissues. In the present study, the HID technique was therefore adapted for post-embedding staining. HID staining was performed on thin sections of chemically and cryofixed kidney and growth plate cartilage, embedded in Epon and various acrylic-based resins. All resins and most tissue preparation conditions allowed post-embedding staining with HID, albeit to variable degrees. However, no significant cytochemical reaction was obtained with tissue sections of osmicated kidney embedded in Epon. Profile views of re-embedded sections showed that large stain deposits were usually restricted to the surface, whereas small ones were observed throughout the entire thickness of the section. The staining pattern was essentially similar between chemically fixed and cryofixed specimens. In the glomerulus, stain deposits were mainly seen over the free surface of podocyte foot processes and over the lamina rara externa. The pericellular cartilage matrix of chemically fixed specimens often appeared as condensed elements, usually stained with large deposits. In cryofixed tissues this matrix formed a meshwork composed of thin, extended filamentous structures, many of which showed linear arrays of smaller stain deposits. The data presented here indicate that post-embedding HID-TCH-SP staining can be successfully performed on thin sections of tissues embedded in various resins and, as a result, can be further adapted to cryo-prepared specimens to give a high resolution localization of sulfated glycoconjugates in tissues with optimal molecular preservation.     

Post-embedding staining of rat gastric mucous cells with lectins

Summary The mucous cells of the rat stomach were stained with lectins by two post-embedding staining methods for electron microscopy. The mucous granules of surface mucous cells and foveolar mucous cells were stained weakly by Ricinus communis agglutinin-ferritin and wheat germ agglutinin-ferritin. The mucous granules of mucous neck cells were stained by concanavalin A-ferritin, Ricinus communis agglutinin-ferritin and wheat germ agglutinin-ferritin. The mucous granules of pyloric gland cells showed an affinity for wheat germ agglutinin-ferritin and concanavalin A-ferritin, while Ricinuscommunis agglutinin-ferritin only slightly stained the granules. The granules of mucous neck cells and pyloric gland cells were also stained by the concanavalin A-horseradish peroxidase-colloidal gold method, but the granules of surface and foveolar mucous cells were not stained by this method. Periodic acid oxidation of the sections before the standard concanavalin A-ferritin procedure enhanced the staining of the granules of mucous neck cells and pyloric gland cells slightly. Reduction of the sections after the periodic acid oxidation weakened the staining. Similar results were obtained using the concanavalin A-horseradish peroxidase-colloidal gold method. Though the staining with Ricinus communis agglutinin-ferritin was inhibited by periodic acid oxidation of the sections before staining, the staining with wheat germ agglutinin-ferritin was not inhibited by the oxidation. It is suggested that the paradoxical staining is closely related to the position of the concanavalin A-binding sugar residues in the carbohydrate chains.This work was supported in part by a grant-in-aid (No. 457008) from the Ministry of Education, Science and Culture, Japan and a grant-in-aid for cancer research (55-21) from the Ministry of Health and Welfare, Japan     

Ia antigens in plastic-embedded tissues: a post-embedding immunohistochemical study

The aim of the present study was to establish a plastic embedding technique that makes possible the immunohistochemical demonstration of class II major histocompatibility complex (MHC) antigens (Ia antigens) in undecalcified joint tissues. Therefore a series of fixatives and dehydrating agents was tested for saving Ia immunoreactivity by post-embedding immunostaining of thin sections (2 microns) of rat tissues that had been embedded in glycol methacrylate (GMA), and by comparing with cryostat sections. An indirect immunoperoxidase and the avidin-biotin complex (ABC) technique were used. Combined with fixation by 4% formaldehyde, dehydration with GMA was found to give the best preservation of Ia antigenicity, followed by dehydration with ethylene glycol. The thinness of tissue sections facilitated the association of Ia antigens with different subcellular compartments in distinct cell populations. These various patterns are described.     

Some monoclonal anti-human lymphocyte and class II MHC antigen antibodies do not stain snap frozen Macaca fascicularis lymphocytes or tissue sections.

Experiments were designed to stain snap frozen and fixed Macaca fascicularis peripheral blood mononuclear cell (PBMC) preparations and colon sections assuming that monoclonal mouse anti-human lymphocyte and class II major histocompatibility complex antigen antibodies would cross-react with the monkey counterparts to the human antigens. Most of the monoclonals used in this study did not stain the monkey frozen and fixed tissues in immunoenzymatic protocols despite adequate staining of control human tissues. The fact that snap frozen Macaca fascicularis PBMCs and tissue sections were not always stained is of practical importance for experimental design in Macaca fascicularis using anti-human reagents.     

Effects of prolonged water washing of tissue samples fixed in formalin on histological staining

The effects of prolonged water washing after fixation for 48 h in 10% (v/v) phosphate-buffered neutral formalin on the quality of representative histological staining methods were evaluated using samples of liver, kidney, spleen and thymus collected from three male Crl:CD(SD)(IGS) rats and one male beagle dog. Because door-to-door courier services in Japan prohibit handling formalin, our goal was to confirm that formalin fixed wet tissue samples could be stored in tap water rather than formalin during transportation of the samples without decreasing the quality of their staining or immunohistochemistry. Each tissue sample was allocated randomly to one of three groups: 12 min, 3 days and 7 days of washing in running tap water; samples then were routinely embedded in paraffin and sectioned. The sections were stained with hematoxylin and eosin, periodic acid-Schiff, azan, and the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method. Immunohistochemical staining for Factor VIII, ED-1 and CD3 also was assessed. Prolonged water washing for up to 7 days did not affect the morphology or stainability by standard histological methods, or the intensity and frequency of positive reactions using the TUNEL method. Only immunohistochemical staining of Factor VIII was altered in both the rat and dog sections after 7 days of water washing. The intensity of positive reactions of Factor VIII immunohistochemistry after 7 days water washing was still strong enough to detect microscopically. Therefore, prolonged water washing for up to 7 days after formalin fixation does not have seriously detrimental effects on the quality and characteristics of paraffin sections stained by various methods, including immunohistochemistry.     

Standardization of various applications of methacrylate embedding and silver methenamine for light and electron microscopy immunocytochemistry

The use of butyl-methyl-methacrylate embedding and the application of the silver methenamine (SM) method as a poststaining of the immunoperoxidase-DAB (IP) procedure led to the standardization of several useful methods for the visualization of tissue antigens at the light and electron microscope level. These procedures included: 1) Standardization of the actual methacrylate embedding; 2) The IP-SM method with and without periodic acid oxidation, which provided 100% intensification of the IP staining; 3) The IP-SM method made it possible to stain semithin sections (0.5 micron), and this in turn, permitted a) clear visualization under the light microscope of the intracellular distribution of antigens and, b) staining, in several adjacent sections, of roughly the same cytoplasmic region of the same cell with different primary antisera; 4) a double immunostaining whereby the first antigen in the sequence was revealed by the IP-SM method and the second by the IP procedure; 5) standardization of the IP and the IP-SM methods for post-embedding staining of ultrathin methacrylate sections. The combined application of methacrylate embedding and the IP-SM, and the use of an appropriate fixative, resulted in an ultrastructural immunocytochemical procedure characterized by a good immunoreactivity of the tissue sections, a strong and selective immunoreaction and a well preserved ultrastructure.     

New sensitive light microscopical detection of colloidal gold on ultrathin sections by reflection contrast microscopy

Summary One simple post-embedding method for combined light- and electron microscopy is presented. Different types of antigens in normal rat and mouse kidneys as well as in tissues from cases of experimental induced nephritis were stained after Lowicryl K4M embedding by an immunogold (silver) method. The (silver-enhanced) gold particles were visualized by light microscopy, e.g. bright-field (BFM)- and reflection contrast (RCM) microscopy, as well as by electron microscopy. The potentials of RCM visualization in this field were investigated, resulting in the successful detection of colloidal gold (15 nm) particles, or silver enhanced gold particles, on ultrathin sections. Furthermore, an increased detection sensitivity of RCM compared with BFM together with an increase in the sensitivity of the immunostaining by RCM visualization was found. The different ways to use RCM, alone or in combination with bright-field- or phase contrast microscopy for visualization of plastic sections varying in thickness, type of plastic and staining, are discussed.     

Tape transfer sectioning of tissue microarrays introduces nonspecific immunohistochemical staining artifacts

Abstract

Tissue microarrays place tens to hundreds of formalin fixed, paraffin embedded tissue cores into a paraffin block in a systematic grid pattern that permits their simultaneous evaluation in a single section. The fragmented nature of the tissue cores often makes sectioning of tissue microarrays difficult so that the resulting disks of tissue lose their shape, fracture or fall out of the paraffin section altogether. We have evaluated an alternative sectioning protocol for stabilizing the tissue microarray surface by placing an adhesive tape “window” over the face of the paraffin block prior to sectioning. Once sectioned, the tape/sections are transferred directly onto coated microscope slides, thereby avoiding routine floating of sections on a water bath. After sectioning with either the tape transfer or standard protocols, slides were stained either using hematoxylin and eosin or immunohistochemistry using antibodies to S-100 protein and the tissue specific antigens, keratin (AE1/3) and the leukocyte common antigen CD45. We found that the tape method produced thicker sections that were darker and more densely packed with loss of tissue definition compared to sections prepared using water bath flotation. Quantitative image analysis of immunohistochemical staining demonstrated that the tape method produced a higher incidence of nonspecific staining, which raised the potential for false positive staining.     

Buffered Romanowsky-Giemsa method for formalin fixed,paraffin embedded sections: taming a traditional stain

Romanowsky-Giemsa (RG) stains were devised during the 19th century for identifying plasmodia parasites in blood smears. Later, RG stains became standard procedures for hematology and cytology. Numerous attempts have been made to apply RG staining to formalin-fixed paraffin-embedded tissue sections, with varied success. Most published work on this topic described RG staining methods in which sections were overstained, then subjected to acid differentiation; unfortunately, the differentiation step often caused inconsistent staining outcomes. If staining is performed under optimal conditions with control of dye concentration, pH, solution temperature and staining time, no differentiation is required. We used RG and 0.002 M buffer, pH 42, for staining and washing sections. All steps were performed at room temperature. After staining and air drying, sections were washed in 96?100% ethanol to remove extraneous stain. Finally, sections were washed in xylene and mounted using DPX. Staining results were similar to routine hemalum and eosin (H &; E) staining. Nuclei were blue; intensity depended largely on chromatin density. RNA-rich sites were purple. Collagen fibers, keratin, muscle cells, erythrocytes and white matter of the central nervous system were stained pinkish and reddish hues. Cartilage matrix, mast cell granules and areas of myxomatous degeneration were purple. Sulfate-rich mucins were stained pale blue, while those lacking sulfate groups were unstained. Deposits of hemosiderin, lipofuscin and melanin were greenish, and calcium deposits were blue. Helicobacter pylori bacteria were violet to purple. The advantages of the method are its close similarity to H &; E staining and technical simplicity. Hemosiderin, H. pylori, mast cell granules, melanin and specific granules of different hematopoietic cells, which are invisible or barely distinguishable by H &; E staining, are visualized. Other advantages over previous RG stains include shorter staining time and avoidance of acetone.     

New sensitive light microscopical detection of colloidal gold on ultrathin sections by reflection contrast microscopy. Combination of reflection contrast and electron microscopy in post-embedding immunogold histochemistry.

One simple post-embedding method for combined light- and electron microscopy is presented. Different types of antigens in normal rat and mouse kidneys as well as in tissues from cases of experimental induced nephritis were stained after Lowicryl K4M embedding by an immunogold (silver) method. The (silver-enhanced) gold particles were visualized by light microscopy, e.g. bright-field (BFM)- and reflection contrast (RCM) microscopy, as well as by electron microscopy. The potentials of RCM visualization in this field were investigated, resulting in the successful detection of colloidal gold (15 nm) particles, or silver enhanced gold particles, on ultrathin sections. Furthermore, an increased detection sensitivity of RCM compared with BFM together with an increase in the sensitivity of the immunostaining by RCM visualization was found. The different ways to use RCM, alone or in combination with bright-field- or phase contrast microscopy for visualization of plastic sections varying in thickness, type of plastic and staining, are discussed.     

The ultrastructure of anionic sites in rat articular cartilage as revealed by different preparation methods and polyethyleneimine staining

The ultrastructure of anionic sites in the middle layer of rat articular cartilages was studied by two methods, the quick-freezing and deep-etching method, and the quick-freezing and freeze-substitution method. The anionic sites were visualized with a cationic tracer, polyethyleneimine. They were also compared with those revealed in tissues subjected to conventional fixation, such as pre-embedding or post-embedding. With the deep-etching method, three-dimensional meshwork structures were observed more clearly in the extracellular matrix compared with those seen in conventional ultrathin sections. In combination with polyethyleneimine staining, in which no chemical contrast was needed for visualization of anionic sites, numerous stained particles were detected around filaments in the extracellular matrix, indicating that they were anionic sites consisting mainly of proteoglycans. With the pre-embedding method and polyethyleneimine staining, the shapes of aggregated stained particles varied with different preparation procedures, including chemical fixation and contrasting. The fine meshworks were also observed with the post-embedding method and polyethyleneimine staining. It is suggested that such images of anionic sites, as revealed by the deep-etching method and the post-embedding polyethyleneimine-staining method with low-temperature dehydration, are probably closer to native states than those revealed by the conventional pre-embedding polyethyleneimine-staining method. © 1998 Chapman & Hall     

Correlative immunofluorescence and electron microscopy on the same section of epon-embedded material

Semithick (0.25-0.50 micron) sections, cut from cells stained with fluorescein isothiocyanate (FITC)-conjugated antibodies prior to embedding in Epon, show high resolution patterns of immunofluorescence against a background void of autofluorescence. These same sections can then be viewed, after uranyl and lead staining, in the electron microscope. We clearly establish the specificity of this same-section correlative immunofluorescence-electron microscopy approach by showing that the immunofluorescent patterns observed in such sections of cells, stained prior to embedding for the indirect immunofluorescent localization of tubulin, follows the distribution of microtubules within the same sections as determined by electron microscopy. We then use this method to demonstrate for the first time that the 57 kD core protein of wound tumor virus is associated, at the ultrastructural level, with two distinct cellular inclusions in virally infected AC-20 cells. In some instances the fidelity in the correlation between the distribution of immunofluorescently labeled antigens and the ultrastructure in the same section eliminates the need to employ more complex procedures for labeling antigens for ultrastructural detection. This technique, therefore, provides a rapid and simple first approach to many problems that require the ultrastructural localization of specific antigens.