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.     

A double-immunostaining procedure using colloidal gold, ferritin, and peroxidase as markers for simultaneous detection of two hypophysial hormones with the electron microscope

For simultaneous ultrastructural localization of intracellular peptides, protein A-gold techniques or immuno-gold techniques have generally been applied. The present study reports a double immunostaining procedure for simultaneous visualization of two hypophysial hormones (prolactin and corticotropin) on a single ultrathin section of the pars distalis of an amphibian. Prolactin and corticotropin antisera were respectively raised in guinea pigs and rabbits and were applied simultaneously to ultrathin sections. Antigenic binding sites were detected under the electron microscope using differently labeled species-specific secondary antisera raised in goats or sheep. Three labels (gold particles, ferritin, peroxidase) were checked for double labeling. The combinations investigated were: 1) two gold preparations or IgG-gold labeled with different-sized gold particles; 2)IgG-gold and IgG-ferritin; 3) IgG-gold and IgG-PAP (peroxidase-antiperoxidase). The double-immunostaining procedures described here have proved useful in the simultaneous ultrastructural localizations of two intracellular antigens on a single tissue section. These procedures constitute a basis for the development of triple immunostaining methods.     

Ultrastructural localisation of oxytocin and neurophysin in the ovine corpus luteum

Summary Polyclonal rabbit antisera raised against oxytocin, bovine neurophysin I and vasopressin were used, together with an immunogold complex, to localise the peptides in ultrathin sections of ovine corpus luteum. The only organelle which consistently showed gold labelling was the secretory granule of the large luteal cell. In non-consecutive sections of the same large luteal cell all the granules showed a similar level of labelling after oxytocin or neurophysin I antisera: however no immunolabelling was detected for vasopressin. Oxytocin and neurophysin seem to be rapidly lost after secretion since exocytosed granule cores showed no labelling above background levels.     

Immunogold staining procedure for the localisation of regulatory peptides

The use of protein A- and IgG-conjugated colloidal gold staining methods for the immuno-localisation of peptide hormones and neurotransmitters at light- and electron microscope level are described and discussed. Bright-field and dark-ground illumination modes have been used to visualise the gold-labelled antigenic sites at the light microscope level. Immunogold staining procedures at the ultrastructural level using region-specific antisera have been adopted to localise specific molecular forms of peptides including gastrin (G17 and G34), glucagon and pro-glucagon, insulin and pro-insulin, in normal tissue and in tumours of the gastroenteropancreatic system. Similar methods have been used to demonstrate the heterogeneity of p-type nerves in the enteric nervous system. Vasoactive intestinal polypeptide (VIP) has been localised to granular sites (mean +/- S.D. granule diameter = 98 +/- 19 nm) in nerve terminals of the enteric plexuses and in tumour cells of diarrhoeogenic VIP-producing neoplasias (mean +/- S.D. granule diameter = 126 +/- 37 nm) using immunogold procedures applied to ultraviolet-cured ultrathin sections. Co-localisation of amines and peptides in carotid body type I cells and in chromaffin cells of normal adrenal medulla and phaeochromocytomas has also been demonstrated. Advantages of the immunogold procedures over alternative immunocytochemical techniques are discussed.     

An investigation of optimal gold particle size for immunohistological immunogold and immunogold-silver staining to be viewed by polarized incident light (EPI polarization) microscopy

We investigated the optimal gold particle size for use with polarized incident light (epi polarization) microscopy with immunogold immunohistological preparation in both immunogold indirect (IGS) and silver-enhanced immunogold-silver staining (IGSS) techniques. A range of gold particle sizes from 5 nm-40 nm was used along with tissue of known immunoreactivity with a well-characterized primary monoclonal antibody. Checkerboard titrations were carried out for each technique and for each particle size. The preparations were viewed using a standard polarized incident light microscope and assessed in a semi-quantitative manner. Adequate visualization of gold particles was achieved using the indirect staining method only with a particle size of 40 nm. With silver enhancement (IGSS), particles of all sizes were clearly seen. However, 5-nm particles were considered optimal for this method because of reduced background staining, high titration of antisera possible, and crisp localization of the visual signal.     

Analysis of the apparent heterogeneity of specific heart granules in rat atrial myocytes; An ultrastructural study including immunocytochemistry

Summary Ultrastructural analysis of specific heart granules in the left atrial myocardium of the rat shows that the so-called A and B types are not different populations but represent varying planes of section through a uniform set of organelles. Histograms of the apparent diameters of granules in individual animals establish that they are unimodal and tilt series of micrographs through selected granules show that the appearances of A and B profiles are interchangeable during the tilting manoeuvre. Moreover, serial sections reveal that equatorial sections through a granule yield the typical appearance of A profiles while peripheral sections through the same granule yield a B type appearance. Immunocytochemical studies were conducted on acrylic resin-embedded specimens using antiserum raised to ANP-28 and visualized by labelling with protein-A gold. A type profiles are more heavily decorated with gold particles than B profiles, a feature which could be partially explained if there is a higher concentration of peptide at the centre of the granule core. Some peripherally sectioned caps of granules which project B type profiles do not exhibit immunoreactivity because their orientation within the thickness of the section prevents access of the antiserum but, after resectioning at right angles to the original plane, their ANP content is revealed by a second phase of immunogold labelling.     

Co-localization of chromogranin A and B,secretogranin II and neuropeptide Y in chromaffin granules of rat adrenal medulla studied by electron microscopic immunocytochemistry

Summary The co-localization of various antigens in rat chromaffin granules was investigated by the immunogold staining procedure. In ultrathin serial sections staining of chromaffin granules was obtained with antisera against chromogranin A, chromogranin B, secretogranin II and neuropeptide Y. These results indicated that these antigens are costored within chromaffin granules. To further corroborate this point a double immunogold staining procedure was used. This method unequivocally established that chromogranin A, chromogranin B, secretogranin II and neuropeptide Y are co-localized in the same chromaffin granules. These results are relevant for studies demonstrating changes in the level of these peptides in adrenal medulla. The co-localization makes it likely that such changes lead to a different relative composition of the secretory quanta of chromaffin granules.     

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.     

Comparison of detecting sensitivities of different sizes of gold particles with electron-microscopic immunogold staining using atrial natriuretic peptide in rat atria as a model

The detecting sensitivities of different-sized gold particles were compared in the localization of atrial natriuretic peptide (ANP) in rat atria. The secondary antibodies were goat antirabbit labeled with 5, 15, 30, or 40 nm colloidal gold diluted 1:2 to 1:100 in Tris buffer. The relative quantity of alpha-ANP immunoreactivity in specific granules was determined by subtracting the number of gold particles in 1 micron 2 nongranule area from that in 1 micron 2 granule area measured with a computerized image analyzer. The optimal dilution that achieved the maximal contrast between specific and background label was influenced by the particle size. Optimal dilutions were 1:80, 1:30, 1:20, and 1:5 for 5, 15, 30, and 40 nm gold, respectively. At optimal dilutions, the maximal detecting sensitivity (MDS) was in inverse proportion to the gold particle size; however, this relationship is not entirely linear. The ratio among the MDSs of 5, 15, 30, and 40 nm gold particles was approximately 34:9:3:2. A double immunogold staining was performed to localize alpha- and beta-ANPs with 15 and 5 nm gold, respectively. Both antigens were detected in the same granules. If the ratios established from the single staining data were used, the ratio between the alpha- and the beta-ANP antigens in the same granules was approximately 2.8:1. The data obtained in this study provide a useful reference for applications of immunogold electron microscopy in a quantitative manner, particularly for double immunogold labeling.     

Ultrastructural immunocytochemistry of glia cells. Double labeling studies using LR White embedding and colloidal gold

The introduction of acrylate resins (Lowicryl K4M, 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 II antigens by pre-embedding method then followed by post-embedding IGS for the other intracytoplasmic antigens.     

Intragranular colocalization of immunoreactive methionine-enkephalin and oxytocin within the nerve terminals in the posterior pituitary

To determine differential tissue antigens in the same section immunocytochemically using the electron microscope, the neurohypophysis was examined following the application of a freeze-drying tissue preparation and staining with the protein A-colloidal gold-antibody complex method (Hisano S, Adachi T, Daikoku S: J Histochem Cytochem 32:705, 1984). At the light microscopic level, colocalized immunostaining for methionine-enkephalin (ENK) and oxytocin (OXT) was found in the rat neurohypophysis under different physiological states. Small pieces of the neurohypophysial tissue were frozen and dried. The dried tissue was fixed with paraformaldehyde vapor and embedded. The ultrathin sections were stained with the antibody for ENK coupled with protein A-small colloidal gold, and antibody for OXT or vasopressin (VP) conjugated with protein A-large colloidal gold. The ultrastructures of the nerve terminals were well preserved and showed many membrane-limited secretory granules. It was possible to identify both OXT- and VP-containing nerve terminals as their secretory granules were differentially labeled with protein A-colloidal gold anti-OXT or anti-VP complex, respectively. The secretory granules, which were labeled with large gold particles for OXT, also carry small gold particles. It is evident that ENK coexists with OXT in the same granules.     

Detection of Cell Surface Antigens in Tissue Sections by Means of Pre-Embedding Immunogold Staining

The immunogold technique has been used in electron microscopy to detect cytoplasmic and extracellular antigens by postembedding techniques. It has also been used to detect plasma-membrane-associated molecules on suspended cells and, recently, to visualise cell surface antigens in ultrathin sections of Lowicryl embedded specimens. In the present study, cell surface antigens of rat kidney and human skin were identified in tissue sections by using pre-embedding immunogold labeling. Brush border microvillar antigens and dermal lymphocyte antigens both bound numerous gold particles. The immunogold staining described here has the advantage over immunoperoxidase procedures that it is not subject to diffusion or reabsorption artifacts, and allows estimation of the antigen density on labeled cells. Furthermore, this pre-embedding immunogold technique is ideally suited to detecting cell surface-associated antigens since it preserves antigenicity, allows gold particle penetration and enhances cell membrane profiles.     

Detection of cell surface antigens in tissue sections by means of pre-embedding immunogold staining

The immunogold technique has been used in electron microscopy to detect cytoplasmic and extracellular antigens by postembedding techniques. It has also been used to detect plasma-membrane-associated molecules on suspended cells and, recently, to visualize cell surface antigens in ultrathin sections of Lowicryl embedded specimens. In the present study, cell surface antigens of rat kidney and human skin were identified in tissue sections by using pre-embedding immunogold labeling. Brush border microvillar antigens and dermal lymphocyte antigens both bound numerous gold particles. The immunogold staining described here has the advantage over immunoperoxidase procedures that is not subject to diffusion or reabsorption artifacts, and allows estimation of the antigen density on labeled cells. Furthermore, this pre-embedding immunogold technique is ideally suited to detecting cell surface-associated antigens since it preserves antigenicity, allows gold particle penetration and enhances cell membrane profiles.     

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     

Sequential use of the PAP and immunogold staining methods for the light microscopical double staining of tissue antigens: Its application to the study of regulatory peptides in the gut

Double immunoperoxidase staining using different couplers can give various combinations of colours on a single tissue section to achieve a comparable picture of different antigens. However, the colour combinations achieved to date are not entirely satisfactory.A double immunostaining procedure is introduced here, combining the peroxidase anti-peroxidase (PAP) and immunogold staining (IGS) methods. The IGS method is a new, simple, sensitive and reliable approach to immunostaining at the light microscopic level. It was carried out in three ways. Firstly, a two-step method was used in which the second layer was goat anti-rabbit IgG adsorbed onto gold particles (GAR/Au20). Secondly, a three-step method was employed where the second layer was unlabelled goat anti-rabbit IgG and the third layer was a rabbit antibody to peroxidase adsorbed onto the gold particles (RAP/Au20) and acting as a gold-labelled IgG antigen. The third method combined the first two methods using GAR/Au20 as the second layer and RAP/Au20 as the third layer which increased the amount of bound gold and enhanced the red colour, providing a better picture.The use of gold-labelled antibodies in double immunostaining has great potential value for many studies including that of the diffuse neuroendocrine system of the gut.     

A multiple-staining ultrastructural procedure simultaneously detecting three membrane antigens on suspended cells by monoclonal antibodies and preembedding immunogold labelling

Summary A triple ultrastructural immunogold staining method for the simultaneous demonstration of three surface antigens of peripheral blood mononuclear cells at the electron microscope level is described. A six-step pre-embedding immunoelectron microscopy procedure was developed, using commercially available reagents. The CD11b antigen was first detected, through a two-step (indirect) method with 40 nm-sized gold particles; after a blocking step, the HLA-DR surface antigen was subsequently detected, through a two-step (biotin-streptavidin) method with 20 nm-sized gold particles; the CD4 antigen was finally detected, through a one-step (direct) method, using 5 nm-sized gold particles. Electron microscopic examination revealed firstly the presence of a triple-labelled cell subpopulation, which showed gold granules of the three sizes simultaneously decorating the cell membrane. Thus, the cells of such a subset simultaneously expressed the three antigens investigated. In contrast, either gold particles of only one size or no gold particles were observed on the cell surface of other subpopulations. This technique is a model demonstrating the importance of varying the size of particles in pre-embedding gold immunoelectron microscopy for a better analysis of the expression of surface antigens in isolated cells.     

Localization and quantification of hormones, ligands, and mRNA with affinity-gold probes

The application of immunogold techniques to localize pituitary hormones produces label that can be quantified and correlated with different secretory states. This report focuses on three major applications of the technology. In the first set of studies, immunogold labels for adrenocorticotropin (ACTH) or luteinizing hormone (LH beta) and follicle-stimulating hormone (FSH beta) were applied to ultrathin sections of pituitaries from adrenalectomized rats or from rats in different stages of the estrous cycle. During the first week after adrenalectomy, ACTH cell area increased. The concentration of immunoperoxidase label (amount of label/area of the corticotropes) decreased. Counts of gold markers showed that there were no changes in the concentration of antigens per granule. Three weeks after adrenalectomy, the amount of immunoperoxidase label increased along with the concentration of that label. The concentration of gold label for ACTH on granules also increased. All changes correlated well with increases in serum ACTH stimulated by adrenalectomy. In the studies of cycling rats, gonadotropes showed increases in the number of gold markers for LH beta or FSH beta per granule area just before an elevation in serum levels. There were also increases in the proportion of granules that contained only LH beta or FSH beta (monohormonal) before the rise in secretion. Thus, nonparallel release of gonadotropins might be attributed to changes in the ratio of gonadotropins packaged per granule. In the second series of studies, avidin-gold labels were used to identify sites of binding of biotinylated ligands. These studies illustrate and quantify binding by biotinylated gonadotropin-releasing hormone (GnRH) to ovarian or pituitary target cells. Triple-labeling protocols (avidin-peroxidase followed by immunogold) show that the target cells in the pituitary contain gonadotropins. In the third set of studies, avidin gold or avidin peroxidase was used to label sites of hybridization of a biotinylated cRNA probe to gonadotropin beta subunit mRNA. The sites of hybridization appear on rough endoplasmic reticulum; however, further work is needed to improve cell ultrastructure and perserve antigens. Triple-labeling protocols (avidin-peroxidase followed by immunogold) show the feasibility of the technique as well as the need for further refinement. To summarize, these studies describe multiple applications of gold labels for the localization of antigens, ligands, and mRNA. The labels are sensitive for detection of antigens and ligands and easily quantified. Quantitative analyses show changes in concentration of gold label that correlate well with secretory states.     

Simultaneous localization of six antigens in single sections of transgenic mouse intestine using a combination of light and fluorescence microscopy.

To study the geographic differentiation of the intestinal epithelium and to understand the complex lineage relationships of its cell populations, it is often necessary to visualize the protein products of multiple genes in sections prepared from different positions along the duodenal-to-colonic and/or crypt-to-villus axes. Multilabel fluorescence or brightfield immunohistochemical techniques have previously been used for this purpose. However, the number of antigens that can be identified on single sections is limited in fluorescence microscopy by the number of fluorophores with non-overlapping absorption and emission characteristics, in brightfield microscopy by the number of visually distinguishable chromogens, and in both methods by the availability of primary antisera raised in multiple species. We have now used a combination of light and fluorescence microscopic techniques to increase the number of antigens that can be detected in a single section to six. Sections were sequentially stained using immunogold with silver intensification, peroxidase-antiperoxidase with diaminobenzidine chromogen, and peroxidase-anti-peroxidase with alpha-naphthol/basic dye as chromogen, followed by simultaneous fluorescent detection with fluorescein, 7-amino-4-methylcoumarin-3-acetic acid, and beta-phycoerythrin. This method enables up to four separate antigens to be visualized within a single cell and two additional antigens to be detected in unrelated cells. The technique is illustrated by examining the cellular patterns of expression of liver fatty acid binding protein/human growth hormone fusion genes in the intestinal epithelium of adult transgenic mice. It should be generally applicable to other experimental systems that require localization of multiple antigens in single tissue sections.     

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.     

Intragranular processing of pro-opiomelanocortin in the intermediate cells of the rat pituitary glands. A quantitative immunocytochemical approach

Quantitative immunocytochemical studies were done by using the immunogold technique on sections of the intermediate lobe of rat pituitary. Antibodies raised (in rabbits) against the precursor proteins pro-opiomelanocortin (POMC) and ACTH were used. The results clearly indicate that the immature granules are the major site of POMC, as their antigenic density (gold beads/micron2) was almost 3 times as high as that of ACTH. In the mature granules, the antigenic density of ACTH was increased by 2.7-fold compared with the immature granules. Using a computer-assisted method, it was possible to categorize the granules' antigenic density according to their size. Using this approach it was found that the antigenic density of POMC remained constant in all mature granules of varied sizes, whereas the antigenic density of ACTH decreased with increasing granule size. The relationship between granule size, degree of maturation, and antigenic density is discussed.