Post-embedding Staining Research Articles

Introductory remarks on glycoconjugate histochemistry

In these opening remarks, many typical methods of demonstrating glycoconjugates were presented. Several such classical demonstration methods at the electron microscopic level have been revived to achieve a sufficient electron density, which makes it possible to use these methods routinely at that level. To detect sugar residues at the electron microscopic level, labeled lectins were used by means of preembedding and postembedding staining. Combining these methods, sugar residues in relation to cell organellae have been successfully detected. Many such examples were reported in this presentation.

Immunoelectron microscopic localization of HNK-1 in the embryonic rat heart.

To confirm the role of HNK-1 in conduction tissue, the ultrastructural localization of monoclonal antibody HNK-1 was analyzed in developing rat hearts at embryonal day 14.5 by immunoelectron microscopic labeling procedures with post-embedding immunogold staining. Tissue sections in different planes containing the sino-atrial (SA) node, atrio-ventricular (AV) node and His bundle were used to demonstrate HNK-1. Immunogold labeling was detected on the cell surfaces and in the extracellular matrices of cells that had features common to conduction tissue cells. Non-specialized contractile myocytes were not labeled by this antibody. Furthermore, immunogold labeling was more prominent in wide intracellular spaces than in narrow intercellular spaces, and rarely observed in cell-cell contact regions. The cell surfaces and extracellular matrices of mesenchymal cells in the endocardial cushion, which contacts the His bundle, were also positive, suggesting the involvement of tract formation to the AV node. These findings may indicate that HNK-1 plays an important role in cell-cell adhesion processes both temporally and spatially in the developing conduction tissue. It was concluded, therefore, that HNK-1 is a suitable marker of the embryonic heart conduction system and might be useful in analyzing anomalous conduction systems, as in congenital heart disease.

Dual Electron Microscopic Localization of Mitochondrial Creatine Kinase in Brain Mitochondria

Mitochondrial creatine kinase in brain mitochondria appears to be located at two different intramitochondrial sites. By using immunogold-labeling techniques, a peripheral immunoreactivity was localized between the two boundary membranes, while an additional, central immunoreactivity was found at the crista surface. The peripheral enzyme was accessible to the antibodies after treatment of the brain mitochondria with 100-300 micrograms digitonin/mg mitochondrial protein, which left 75% of the activity bound to the membranes. Electron microscopic analyses revealed that 43% of the labeled, peripheral creatine kinase was bound at those places where outer membrane vesicles remained attached to the inner envelope membrane, suggesting that the enzyme is involved in contact formation between outer and inner mitochondrial membranes. Postembedding staining of mitochondria on thin sections of brain tissue or in the isolated state led to the observation of a second location of creatine kinase inside the mitochondria, along the cristae, which was not accessible to the antibodies in isolated, digitonin-treated mitochondria.

Ultrastructural evidence for intra‐ and extranuclear projections of GABAergic neurons of the suprachiasmatic nucleus

GABAergic projections of the suprachiasmatic nucleus (SCN) were demonstrated in a double-labelling ultrastructural study which visualised the efferents of the SCN by PHA-L tracing, diaminobenzidine (DAB) immunocytochemistry, and GABA with immunogold postembedding staining. The results show a strong contralateral projection of the SCN that is partly GABA-containing. In addition, ipsilateral SCN projections to the dorsomedial hypothalamus and periventricular part of the paraventricular nucleus and sub-paraventricular nucleus were shown to contain GABA. The present results indicate that the SCN may utilize this inhibitory neurotransmitter to regulate and organize its own circadian rhythm as well as using GABA to transmit its diurnal information to other regions of the brain.

Colocalization of somatostatin with GABA or glutamate in distinct afferent terminals presynaptic to the Mauthner cell

The presence of somatostatin in afferent fibers impinging on the goldfish Mauthner (M-) cell was determined using immunohistochemical methods, combined with confocal and electron microscopy, and the relationship of this peptide with inhibitory and excitatory terminals was studied. Somatostatin-reactive boutons were present only on the distal part of the M-cell's lateral dendrite. Somatostatin immunoreactivity was observed in typical large myelinated club endings (LMCEs) corresponding to mixed (electrical and chemical) eighth nerve primary afferent fibers. The axoplasm of these fibers contained dense-core vesicles (DCVs) dispersed among round vesicles. We have made a novel finding that the excitatory transmitter glutamate is present in LMCEs. Colocalization of this amino acid with somatostatin was detected in 75% of these endings using postembedding staining with gold particles of various sizes. The other structures labeled by somatostatin antibody were found to be small vesicle boutons (SVBs), which establish symmetrical synapses and contain a population of pleiomorphic vesicles with DCVs scattered among them. Double labeling with antibodies against glutamic acid decarboxylase and GABA allowed the definition of three types of biochemically characterized terminals: [somatostatin-GABA], [GABA], and [somatostatin]. However, the occurrence of DCVs in SVBs stained for GABA alone suggests that neuropeptides other than somatostatin may also coexist with GABA in this class of boutons. The coexistence of somatostatin with both inhibitory and excitatory neurotransmitters acting on the same region of a postsynaptic cell is discussed in relation to the role postulated for this peptide in synaptic plasticity.

Immunocytochemical Localization of Blood Group ABH Antigens in the Human Parotid Gland

The aim of this study was to determine whether the parotid gland, like other salivary glands, produces blood group antigens. A post-embedding immunogold staining (IGS) method for ABH antigens was applied to osmicated and nonosmicated, routinely prepared specimens of normal human parotid glands. A and B antigens were not detected, whereas H antigen was found in acini and ducts of glands from A, B, and O subjects. In osmicated samples, the only sites of H labeling were secretory granules of a few acinar cells and those of most intercalated duct cells. In both cell types, only the pale matrix of the granules was stained. In non-osmicated samples, secretory granules showed the same distribution pattern of H, although gold particles were more abundant, and basolateral membranes of acinar and striated duct cells and the apical vesicles of striated duct cells were also labeled. Our results suggest that small amounts of H antigen are secreted by the parotid gland.

Electron microscopic immunocytochemistry of glutamate‐containing nerve fibers in the taste bud of mudpuppy (Necturus maculosus)

The presence of glutamate immunoreactivity (glu-IR) in the nerve fibers of the mudpuppy taste bud was investigated by electron microscopy. Pre-embedding staining with avidin-biotin-peroxidase complex (ABC) and post-embedding staining with 5 nm colloid gold conjugates were used separately to identify immuno-stained structures. We have found the following: 1) the majority of the nerve fibers innervating the mudpuppy taste bud are unmyelinated; 2) about 85% of nerve fibers located at the base of the taste bud and about 60% of the nerve fibers located between the taste cells show glu-IR by pre-embedding staining; 3) there is a preferential staining of the glu-IR in the nerve fibers of the mudpuppy taste bud; and 4) the distribution of the colloidal gold particles in the nerve fibers is 1.5 to 2 times denser than that of the staining in the connective tissue background or cellular profiles of taste cells. From the distribution and pattern of the nerve fibers obtained in the thick and thin sections, we conclude that the mudpuppy taste bud is innervated by glutamate-containing unmyelinated nerve fibers.

Synaptic relationships between gaba-immunoreactive boutons and primary afferent terminals in the rat cuneate nucleus

The present study examined the synaptic relation between the primary afferent terminals and intrinsic neuronal elements in the rat cuneate nucleus. For this purpose, experimental degeneration after multiple cervicothoracic dorsal rhizotomies or anterograde transport of wheatgerm agglutinin conjugated to horseradish peroxidase were used to identify the primary afferent terminals, while immunogold postembedding staining was employed to identify the GABA-immunoreactive boutons. The combined procedure allowed us to demonstrate a direct synaptic relationship between the primary afferent terminals and GABA-immunoreactive boutons. At least two types of synaptic relation were observed between the primary afferent terminals, identified by their degenerating features or labeled by wheatgerm agglutinin conjugated to horseradish peroxidase, and the immunogold-labeled GABA-immunoreactive boutons (i) a GABA-immunoreactive bouton making a simple presynaptic contact with the primary afferent terminal; and (ii) a synaptic glomerular complex in which the centrally located primary afferent terminal was postsynaptic to a GABA-immunoreactive bouton and presynaptic to dendrites closely associated with it; both terminals were sometimes presynaptic to a common dendrite. It is speculated from this study that the incoming impulses from the forelimb area are modulated by the GABA-immunoreactive boutons in the cuneate nucleus of the rat.

GABAergic regulation of noradrenergic spinal projection neurons of the A5 cell group in the rat: An electron microscopic analysis

Recent studies have demonstrated an important contribution of the A5 noradrenergic cell group of the rostral medulla in the regulation of nociceptive messages at the level of the spinal cord. These noradrenergic controls parallel those arising from the serotonin-containing neurons of the nucleus raphe magnus. In the present study, we used postembedding immunogold staining to identify GABA-immunoreactive terminals that synapse upon identified spinally projecting noradrenergic neurons of the A5 cell group in the rat. A5 projection neurons were identified by Fluoro-Gold transport from the spinal cord; sections containing retrogradely labelled cells were then immunoreacted for tyrosine hydroxylase (TH) to identify the catecholamine-containing, presumed noradrenergic, neurons. Double-labelled A5 cells were intracellularly filled with Lucifer Yellow (LY) and then the LY was photo-oxidized to an electron-dense product. Seven intracellularly filled TH-immunoreactive projection neurons were studied with postembedding immunocytochemistry. Each A5 neuron received a significant GABA-immunoreactive terminal input. Out of a pooled total of 151 terminal profiles found in apposition to intracellularly labelled somatic and dendritic profiles, 31 (20.5%) were GABA-immunoreactive. The proportion of GABA-immunoreactive terminals that contacted somatic profiles (12/72; 17%) was similar to the proportion that contacted TH-labelled dendritic profiles (19/79; 24%). There was a discernible synaptic specialization in about 50% of the labelled terminals that contacted the TH projection neuron. Both symmetric and asymmetric synaptic specializations were found. Labelled terminals contained round or pleiomorphic vesicles, but not flat vesicles; many also contained dense-core vesicles. Our results indicate that noradrenergic neurons of the A5 cell group, which contribute to both antinociceptive and cardiovascular controls through their projection to the spinal cord, are regulated by local GABAergic, presumably inhibitory, mechanisms. Whether the initiation of A5 neuron activity results from a lifting of tonic GABAergic inhibitory control, as has been proposed for the neurons of the nucleus raphe magnus, remains to be determined.

Pathological mineralization in a serially passaged cell line from rat pulp.

The ultrastructure of crystal formation in association with dental pulp cells isolated from rat incisor was studied in vitro. A clone, RPC-K, was obtained and incubated with Na-beta-glycerophosphate (BGP). Growing pulp cells showed low alkaline phosphatase (ALP) activity, which began to increase with cell proliferation. Pulp cells formed cell multilayers after day 14 of culture. Mineralized tissues were observed within cell multilayers on day 28 of culture. Vesicular structures were found around degenerate and necrotic cells. Some of these vesicles contained needle-like crystals. Organic structures appeared at the periphery of mineralized tissues with a post-embedding demineralization and staining method. Electron diffraction patterns of the newly formed crystals revealed a pattern consistent with hydroxyapatite (HAP). These findings suggest that the RPC-K cell line might be useful for a model system to investigate pathological mineralization.

Catecholaminergic innervation of oxytocin neurons in the paraventricular nucleus of the rat hypothalamus as revealed by double-labeling immunoelectron microscopy.

The catecholaminergic innervation of neurons that contain oxytocin in the paraventricular nucleus (PVN) of the rat hypothalamus was examined by a combination of methods in the same tissue sections at the electron-microscopic level as follows: (1) Rats were treated with 5-hydroxydopamine (5-OHDA) with peroxidase-antiperoxidase (PAP) staining of sections for oxytocin prior to embedding. (2) Preembedding immunoperoxidase staining with avidin-biotin complexes was used to demonstrate tyrosine hydroxylase (TH) activity, with postembedding staining with immunocolloidal gold for visualization of oxytocin. (3) Prior to embedding, a double-staining technique was used that was based on consecutive staining with silver-gold-intensified PAP complex and 3,3'-diaminobenzidine. We used an antiserum against oxytocin and an antiserum against dopamine-beta-hydroxylase (DBH) for localization of antigens. We found that TH- and DBH-like immunoreactive terminals were distributed throughout the rat hypothalamus and were abundant in all parts of the PVN. Ultrastructural observations revealed 5-OHDA-labeled, TH- or DBH-like immunoreactive axon terminals that contained granular vesicles (70-80 nm in diameter) and small clear synaptic vesicles (30-50 nm in diameter). The terminals appeared at times to be making synapses with cell bodies and with the processes of oxytocin-containing neurosecretory neurons in the PVN. These findings provide morphological evidence for a direct synaptic influence of catecholaminergic elements on the secretory activity of oxytocin-containing neurosecretory neurons in the rat hypothalamic PVN.

A postembedding method using urea-unmasked ultrathin sections for the electron microscopic detection of sulfated glycoconjugates.

Using urea-unmasked ultrathin sections, a postembedding staining method in combination with nitrous acid procedure has been established for the efficient visualization of sulfated glycoconjugates in a series of ultrastructures of three salivary glands of the rat. Pieces of the parotid, submandibular and sublingual glands from adult rats were subjected to glutaraldehyde-paraformaldehyde fixation, rinsed, dehydrated and embedded in LR-White resin. Ultrathin sections were prepared, grid-mounted, incubated in urea (6M) aqueous solution, and then stained with an alcian blue (AB) pH 1.0-phosphotungstic acid (PTA) sequence with or without prior treatment with nitrous acid. The results obtained have shown that sulfated glycoconjugates are involved in such ultrastructures as mast cell granules, basal lamina of arteriolar endothelial cells and intercellular matrix of arteriolar smooth muscle cells.

Electron microscopic immunocytochemical localization of proline-rich proteins in normal mouse parotid salivary glands.

Rabbit polyclonal antibodies against isoproterenol-induced mouse proline-rich proteins (PRPs) were used to localize PRPs in the parotid salivary glands of normal adult BALB/c mice. The antibodies recognized both acidic-type and basic-type PRPs. Immunoblotting experiments revealed that the glands contained an acidic-type and a basic-type PRP. Parotid gland tissue was fixed with Karnosky's fixative and embedded in Lowicryl resin at low temperature. PRPs were localized at the electron microscope level using an indirect post-embedding staining technique with protein A-gold. The secretion granules of the acinar cells were strongly labelled. Pre-absorption of the antibody with purified acidic-type and basic-type PRPs indicated that the basic-type PRP is mainly located at the periphery of the granules but that the acidic-type PRP is more evenly distributed within the granules. Pre-absorption of the antibody with alpha-amylase did not affect the staining pattern, suggesting minimal cross-reactivity. PRPs were also detected within the rough endoplasmic reticulum and the Golgi apparatus of acinar cells, within the granules of the proacinar cells and in the lumena of the ducts, but not within the intercalated or striated duct cell granules.

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.

Lectin cytochemistry in the gastrointestinal tract with special reference to glycosylation in the Golgi apparatus of Brunner's gland cells.

Two hydrophilic, low temperature-embedding resins, Lowicryl K4M and LR White, were compared in lectin cytochemistry. Post-embedding staining of colloidal gold-labeled Griffonia symplicifolia agglutinin II (GSA-II) resulted in staining of the Golgi apparatus and mucous granules of mucous neck cells in the gastric fundic gland, pylorocytes, and Brunner's gland cells embedded in either resin, although it was much easier to make ultra-thin sections with LR White-embedded material than with the other. Post-fixation with uranyl acetate followed by LR White embedding improved general ultrastructure so that lectin binding sites were identified precisely. All examined lectins, soybean agglutinin (SBA), Maclura pomifera agglutinin (MPA), GSA-II, and Ulex europaeus agglutinin I (UEA-I), stained mucous granules and the Golgi apparatus, in which the staining pattern was characteristic of each lectin: cis cisternae were labeled with SBA and MPA, intermediate cisternae with GSA-II, and trans cisternae and mucous granules with SBA, GSA-II, UEA-I, and lightly with MPA. No labeling was observed in the rough endoplasmic reticulum with any lectin. These findings suggest that the Golgi apparatus is the site of O-linked glycosylation and can be divided into at least three distinct compartments with regard to the glycosylation.

Sulfated Glycoconjugates Demonstrated in Combination with High Iron Diamine Thiocarbohydrazide-Silver Proteinate and Silver Acetate Physical Development

Sulfated glycoconjugates in epithelial cells and mesenchymal cells were investigated after staining with high iron diamine-thiocarbohydrazide-silver proteinate. One purpose of the experiment was to apply a new physical developed to the staining. Instead of silver nitrate, silver lactate or silver bromide, we used silver acetate as an ion donor. This new method allowed physical development under normal lighting conditions, and resulted in the reduction of background staining even after amplification. As the developer did not contain gum arabic, troublesome treatment was not necessary. The time required for staining was very short and the electron density of the final reaction product was high and easily identifiable under the electron microscope. Fixing was not necessary. Very small amounts of reactive substance were detectable after physical development. This developmental procedure has been applied to both the preembedding staining and postembedding staining of sulfated glycoconjugates. The results obtained using this method are presented.

Distribution of beta-A4 protein and APP in the vessels of normal and pathological brains: M.Gonzales ∗, S.Davis +, K.Beyreuther #, C.L. Masters ∗. Department of Pathology ∗, University of Melbourne and Neurology +, The Royal Melbourne Hospital, Melbourne, 3050, Australia and Center for Molecular Biology #, University of Heidelberg, Germany

The cell surface and intracellular binding sites of two lectins, wheat germ agglutinin (WGA) and Ricinus communis agglutinin-1 (RCA-1), in the monkey retina were investigated at the ultrastructural level by means of post-embedding staining of Lowicryl K4M-embedded specimens with lectin-colloidal gold complexes. Effects of pretreatment with neuraminidase were also studied. The techniques enabled us to detect lectin binding sites in the various intracellular compartments of photoreceptor cells, retinal pigment epithelium (RPE), and interphotoreceptor matrices. Wheat germ agglutinin, which recognizes N-acetylglucosamine and sialic-acid residues, showed a prominent and uniform binding to the disc membranes of rod outer segments (ROS), rod connecting cilia, interphotoreceptor matrices, and RPE microvilli. The intensity of these binding sites was markedly reduced when thin sections were pretreated with neuraminidase, except the labeling of ROS disc membranes. Cones were labeled sparsely. The staining of phagosomes in the RPE cytoplasm did not change after neuraminidase digestion. RCA-1, which recognizes galactose residues, revealed a moderate binding to the ROS disc membranes, with a noticeably greater intensity of binding to the basal region of ROS disc membranes. With neuraminidase treatment heavier binding with RCA-1 occurred in interphotoreceptor matrices, connecting cilia and RPE microvilli, whereas there was no significant alteration in binding to the ROS disc membranes. Phagosomes in the RPE were also labeled with this lectin. The results suggest the presence of sialic acid and galactose as the constituent carbohydrates of glycoconjugates in the interphotoreceptor matrices and RPE microvilli.

7.11 A new polyclonal monospecific antibody, reactive with neurotensin in glutaraldehyde- and osmiumtetroxide-fixed mammalian brains using pre- as well as postembedding staining protocols

7.11 A new polyclonal monospecific antibody, reactive with neurotensin in glutaraldehyde- and osmiumtetroxide-fixed mammalian brains using pre- as well as postembedding staining protocols

Gold enhancement of gold-labeled probes: gold-intensified staining technique (GIST).

In this report we present a staining method in which gold chloride is used to enhance the size of gold colloids. We show the utility of this technique when used in conjunction with small gold colloids, i.e., 5 nm, 4 nm, and 2.6 nm. Post-embedding staining of epoxy-embedded, gold-labeled mouse LM fibroblasts showed that staining with 0.1% gold chloride facilitated the visualization of the smallest gold colloids.

Antibodies against neuroactive amino acids and neuropeptides. I. A new two-step procedure for their conjugation to carrier proteins and the production of an anti-Met-enkephalin antibody reactive with glutaraldehyde-fixed tissues.

We developed a new two-step procedure to couple haptens to bovine serum albumin (BSA) via glutaraldehyde (GA). After activation of BSA with excess GA and removal of unreacted GA, the hapten was bound to the activated protein in a second step. This two-step procedure is easy to use, the desired molecular ratio of coupled hapten to protein is conveniently adjusted, and no visible precipitation of the conjugate is detected. Using a low peptide concentration, nearly 50% of the inserted haptens are bound to the protein, and unbound expensive peptide can be recovered after Sephadex chromatography. Antisera to neuroactive amino acids (GABA, glycine, and glutamate) and neuropeptides (Met-enkephalin) were prepared by immunization of rabbits with these conjugates. Immunological analysis of immune sera by dot-blot and ELISA techniques and subsequent removal of crossreactivities by solid-phase adsorption yielded monospecific antibodies, which were further purified by affinity chromatography. The immunocytochemical specificities of these purified antibodies were verified in adjacent sections of GA-fixed rat spinal cord. Pre-embedding staining with anti-Met-enkephalin in combination with post-embedding staining for amino acids such as GABA allowed double staining of the two antigens in a single semi-thin section.