Autoradiographic detection of [125I]-secondary antiserum: a sensitive light and electron microscopic labeling method compatible with peroxidase immunocytochemistry for dual localization of neuronal antigens

We examined whether autoradiographic localization of [125I]-antirabbit immunoglobulin (IgG) was suitable for light and electron microscopic detection of a rabbit antiserum to the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), and whether autoradiographic and peroxidase labeling could be combined for simultaneous immunocytochemical identification of TH and neuropeptides in brain. Adult rat brains were fixed by aortic arch perfusion with acrolein and paraformaldehyde. Vibratome sections of the fixed tissues were incubated with various dilutions of TH antiserum followed by [125I]-secondary IgG. These sections were then directly processed for autoradiography or were incubated with rabbit antiserum to substance P (SP) or methionine [Met5]-enkephalin (ME). These latter sections were then processed by the peroxidase-antiperoxidase (PAP) or conjugated peroxidase methods followed by autoradiography. Exposure periods of 12-20 days for light microscopy or 90 days for electron microscopy yielded substantial accumulations of silver grains even at the highest (1:30,000) dilution of TH antiserum. At this dilution, immunoreactivity for TH was virtually nondetectable by PAP and conjugated peroxidase methods. The differential sensitivities of the autoradiographic versus peroxidase methods provided a means for separable identification of rabbit antiserum to TH and to SP or ME. Ultrastructural analysis of the catecholaminergic neurons in the medial nuclei of the solitary tract (NTS) showed selective cytoplasmic localization of silver grains for [125I]-labeling of TH in perikarya, dendrites, and terminals. Within single thin sections prepared for dual labeling, the peroxidase marker for SP and for ME was differentially localized with respect to autoradiographic labeling of TH.     

Localization by immunoelectron microscopy of somatostatin-containing cells in the gastric mucosa of a teleost fish, Perca fluviatilis

Somatostatin-immunoreactive cells were localized on semithin and ultrathin sections of Epon-embedded samples of perch gastric mucosa, classically fixed with aldehydes and osmium tetroxide. On semithin sections, somatostatin cells were identified by using the immunoperoxidase method. The ultrastructural localization of somatostatin immunoreactivity was achieved using the colloidal gold method. Cells showing somatostatin immunoreactivity are found to be scattered among the surface mucous cells and the mucous neck cells. Somatostatin appears to be localized in cytoplasmic granules. Somatostatin-containing cells are identified as the type I cells which were described in a previous ultrastructural study. The present report also points out that tissue samples which have been classically processed for ultrastructural study could be in some cases suitable for immunocytochemical investigations.     

Ultrastructural colocalization of the bioactive mediators 5-hydroxytryptamine and bombesin in endocrine cells of human fetal airways

Summary Endocrine cells in the airway epithelium of human fetal lungs are known to contain an amine, 5-hydroxytryptamine (5HT), and a peptide, bombesin (BOM). These mediators may be involved in regulating smooth muscle and secretory activity in the airways as well as in development of the fetal lung. However, the exact endocrine cell type that contains 5HT and BOM has not been described at the ultrastructural level. This investigation provides immunocytochemical evidence that 5HT and BOM are stored in a single cell type, the P1 cell. Thin sections of airways from human fetal lungs were incubated either in anti-5HT antiserum (diluted 13000) or in anti-BOM antiserum (diluted 1600) and then labeled with affinity purified goat anti-rabbit IgG coupled to 16 nm gold particles. For colocalization, thin sections were incubated on one side to demonstrate 5HT and on the other side to demonstrate BOM. Two different sizes of gold particles (10 and 30 nm) were coupled to IgGs and used for the labeled second antibodies. Controls consisted of absorbing of the primary antiserum with an excess of either 5HT or BOM. 5HT-and BOM-like immunoreactivities were observed in the dense-core vesicles (DCV) of P1 cells, and it was apparent from serial sections that 5HT and BOM labeling was sometimes present in the same P1 cells. Sections labeled for 5HT on one side with large gold particles and for BOM on the other side with small gold particles revealed that 5HT-and BOM-like immunoreactivities were located in the same DCV. Labeling did not occur when the anti-5HT antiserum was absorbed with 5HT or when the anti-BOM antiserum was absorbed with BOM. These results demonstrate that 5HT-and BOM-like immunoreactivity is present in P1 endocrine cells of human fetal lung. Furthermore, a single DCV contains both 5HT and BOM.     

Subcellular localization of serotonin immunoreactivity in rat enterochromaffin cells

Serotonin immunoreactive material was localized to rat enterochromaffin cells (EC cells) at the subcellular level using antibodies to serotonin (5-HT) raised in rabbits. Ultrathin sections from paraformaldehyde fixed plastic embedded tissues were directly labelled with the 5-HT antiserum, using the protein A-gold technique to visualize the immunoreaction. The 5-HT immunoreactivity (5-HT-IR) in the rat gastrointestinal mucosa was exclusively localized to epithelial EC cells with a low background over other epithelial non-enterochromaffin cells. Quantitative evaluation of the immunoreaction revealed that most of the 5-HT-IR in the cytoplasm of EC cells (60%) was located over the dense cores of the secretory granules. However, a significant part of the cytoplasmic 5-HT-IR (40%) was located outside the dense cores of the secretory granules which suggests that different forms of 5-HT storage may exist.     

Light microscopic immunocytochemical demonstration of peroxisomal enzymes in epon sections

A procedure is described for light microscopic immunocytochemical localization of catalase and three enzymes of peroxisomal lipid beta-oxidation: acyl-CoA oxidase, enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase in semi-thin sections of rat liver processed for routine electron microscopy. Satisfactory immunostaining required the removal of the epoxy resin with sodium ethoxide, controlled digestion of deplasticized sections with proteases and, in case of osmiumfixed tissue, bleaching with oxidants. Resin removal was essential for successful immunostaining, and protease treatment enhanced markedly the intensity of the reaction. This study shows that tissues processed for conventional ultrastructural studies can be used for postembedding immunocytochemical demonstration of various peroxisomal enzymes.     

Chromaffinity, uranaffinity and argentaffinity of small granule-containing (SGC) cells in rat superior cervical ganglia.

A systemic examination on the small granule-containing (SGC) cells in rat superior cervical ganglia was conducted by conventional and cytochemical electron microscopy including chromaffin, argentaffin and uranaffin reactions. According to the fine structure of dense cored vesicles (DCVs) in the cytoplasm, three types of small granule-containing (SGC) cells were revealed--Type I: 90-160 nm vesicles with cores of moderate or low electron density; Type II: 130-330 nm vesicles, polymorphic with highly electron dense cores; Type III: elongated vesicles (170 nm x 60 nm) with cores of moderate to low electron density. The majority of SGC cells were the Type I cells (78%) and Type II and III cells made up 13% and 9% of SGC cell population, respectively. Cytochemical results demonstrated that only the Type II cells displayed a positive chromaffin reaction and all three types of SGC cells showed argentaffinity and uranaffinity. The present study is the first to demonstrate the argentaffin reaction at ultrastructural level in SGC cells of sympathetic ganglia. Based on the results of the present study we also concluded that (1) the DCVs of Type II SGC cells contained noradrenaline and (2) biogenic amines and nucleotides (ATPs) coexisted in the DCVs of all three types of SGC cells.     

Immunocytochemical demonstration of peroxisomal enzymes in human kidney biopsies

Peroxisomes are particularly abundant in the proximal tubules of the mammalian kidney. We describe the immunocytochemical localization of catalase and three peroxisomal lipid beta-oxidation enzymes: acyl-CoA oxidase, bifunctional protein (enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase) and 3-ketoacyl-CoA thiolase, in human renal biopsies fixed with glutaraldehyde and embedded in Epon. For light microscopy of semithin sections, satisfactory immunostaining required removal of the resin and controlled proteolytic digestion followed by the indirect immunoperoxidase technique. Brief etching of ultrathin sections with alkoxide followed by the protein A-gold method were used for electron microscopic localization of the enzymes. The immunoreactive peroxisomes were distinctly visualized in proximal tubular epithelial cells with no staining of any other cell organelles. The results establish the presence of catalase and of peroxisomal lipid beta-oxidation system proteins in human kidney. The immunocytochemical procedure described herein provides a simple approach for the investigation of peroxisomal structure and function in human renal biopsies processed for ultrastructural studies.     

Subcellular localization of serotonin immunoreactivity in rat enterochromaffin cells

Summary Serotonin immunoreactive material was localized to rat enterochromaffin cells (EC cells) at the subcellular level using antibodies to serotonin (5-HT) raised in rabbits. Ultrathin sections from paraformaldehyde fixed plastic embedded tissues were directly labelled with the 5-HT antiserum, using the protein A-gold technique to visualize the immunoreaction. The 5-HT immunoreactivity (5-HT-IR) in the rat gastrointestinal mucosa was exclusively localized to epithelial EC cells with a low background over other epithelial non-enterochromaffin cells. Quantitative evaluation of the immunoreaction revealed that most of the 5-HT-IR in the cytoplasm of EC cells (60%) was located over the dense cores of the secretory granules. However, a significant part of the cytoplasmic 5-HT-IR (40%) was located outside the dense cores of the secretory granules which suggests that different forms of 5-HT storage may exist.Supported by grants from the Swedish Medical Research Council (537, 2207, 5220). Göteborgs Läkaresällskap, and The Medical Faculty of Göteborg     

Two-color Fluorescence Labeling in Acrolein-fixed Brain Tissue

Acrolein is a potent fixative that provides both excellent preservation of ultrastructural morphology and retention of antigenicity, thus it is frequently used for immunocytochemical detection of antigens at the electron microscopic level. However, acrolein is not commonly used for fluorescence microscopy because of concerns about possible autofluorescence and destruction of the luminosity of fluorescent dyes. Here we describe a simple protocol that allows fine visualization of two fluorescent markers in 40-μm sections from acrolein-perfused rat brain. Autofluorescence was removed by pretreatment with 1% sodium borohydride for 30 min, and subsequent incubation in a 50% ethanol solution containing 0.3% hydrogen peroxide enhanced fluorescence labeling. Thus, fluorescence labeling can be used for high-quality detection of markers in tissue perfused with acrolein. Furthermore, adjacent acrolein-fixed sections from a single experiment can be processed to produce high-quality results for electron microscopy or fluorescence labeling. (J Histochem Cytochem 58:359–368, 2010)     

Light microscopic immunocytochemical demonstration of peroxisomal enzymes in epon sections

Summary A procedure is described for light microscopic immunocytochemical localization of catalase and three enzymes of peroxisomal lipid -oxidation: acyl-CoA oxidase, enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase in semithin sections of rat liver processed for routine electron microscopy. Satisfactory immunostaining required the removal of the epoxy resin with sodium ethoxide, controlled digestion of deplasticized sections with proteases and, in case of osmiumfixed tissue, bleaching with oxidants. Resin removal was essential for successful immunostaining, and protease treatment enhanced markedly the intensity of the reaction. This study shows that tissues processed for conventional ultrastructural studies can be used for postembedding immunocytochemical demonstration of various peroxisomal enzymes.Supported by the Alexander-von-Humboldt Foundation     

An improved immunocytochemical method for subcellular localization of serotonin in rat enterochromaffin cells

Serotonin-like immunoreactivity (5-HT-LI) has been localized at the ultrastructural level in enterochromaffin (EC) cells of rat gastrointestinal tract. Ultra-thin sections of tissues embedded in epoxy resin were incubated with 5-HT antisera and antibody binding sites were visualized with protein A-gold. Three different antisera were compared and were shown to require different fixation regimens for optimal preservation of 5-HT-LI. For one antiserum, tissues fixed in glutaraldehyde and osmium tetroxide could be used to demonstrate 5-HT-LI in EC cells. Immunocytochemical localization of 5-HT can thus be performed with good ultrastructural preservation of tissues. Quantitative evaluation of the intracellular distribution of 5-HT-LI was performed on EC cells from antrum, duodenum, and proximal colon, fixed in glutaraldehyde only. In all three locations, the majority of the gold particles (90%) in EC cells were localized over the dense core of the secretory granules, while a minor fraction (10%) were localized in parts of the cytoplasm devoid of granules. In EC cells fixed in glutaraldehyde and post-fixed in osmium tetroxide, 5-HT-LI was reduced by about 85%, although intracellular distribution was essentially the same as in cells fixed in glutaraldehyde alone. The results indicate that 5-HT in EC cells is stored mainly in secretory granules, with a small fraction of 5-HT being localized outside the granules.     

Unconventional application of standard light and electron immunocytochemical analysis to aldehyde-fixed, araldite-embedded tissues

A simple procedure for the immunocytochemical analysis of glutaraldehyde/formaldehyde-fixed, Araldite- or Epon-embedded tissues by either light or electron microscopy is presented. Retention of immunoreactive antigen in deplasticized sections was achieved by use of a low concentration of glutaraldehyde in the fixative in combination with a seldom-used plastic solvent. This protocol produced good ultrastructural preservation in tissues and large, high-quality, 2-micrometers thick, plastic-free sections. These semithin sections provided a level of structural and antigenic preservation, image resolution, and labeling intensity that surpassed all other conventional sectioning methods used for immunocytochemistry. The capacity to use a single tissue sample in studies designed for light and electron immunocytochemistry, in conjunction with existing autoradiographic and cytochemical techniques, makes this a very desirable method for routine tissue preparation in research and clinical applications.     

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.     

Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique

The protein A-gold immunocytochemical technique has been modified to allow labeling of cellular antigenic sites on osmium-fixed or postfixed tissues. Several strong oxidizing agents have been found able to restore protein antigenicity on osmicated tissue thin sections. According to the fine structural preservation and intensities of labeling, pretreatment with sodium metaperiodate gave optimal results. Pancreatic secretory proteins (and/or proproteins) as well as insulin (and/or proinsulin) were localized over perfectly preserved rough endoplasmic reticulum (rER), Golgi apparatus, and secretory granules of the corresponding pancreatic cells; carbamyl phosphate synthetase and catalase were revealed over liver mitochondria and peroxisomes, respectively. In addition to the higher resolution in the labeling obtained using osmium-fixed tissues, the present modification confers an additional advantage to the protein A-gold technique by allowing labeling on tissues processed for routine electron microscopy.     

Mycoplasma pneumoniae cytadherence phase-variable protein HMW3 is a component of the attachment organelle.

The subcellular location of the phase-variable cytadherence-accessory protein HMW3 in Mycoplasma pneumoniae has been examined by biochemical and immunoelectron microscopic techniques. Analysis by Western blot (immunoblot) with HMW3-specific antiserum established the presence of this protein within the M. pneumoniae Triton X-100-insoluble fraction or triton shell. Immunogold labeling of Triton-extracted mycoplasmas with affinity-purified antibodies localized HMW3 to the terminal knob on the rodlike extensions of the triton shell, a location that would correspond to the adherence organelle in whole mycoplasmas. Treatment of triton shells with KI resulted in the selective removal of the adherence-accessory proteins HMW1 to HMW4. Analysis of these triton shells by transmission electron microscopy revealed dramatic ultrastructural changes in the filamentous network and core structure. Immunogold labeling of KI-extracted shells reflected the removal of HMW3 from the disrupted tip structure. An examination of ultrathin sections of wild-type cells by transmission electron microscopy following labeling with HMW3-specific antibodies provided further evidence for the nonrandom distribution of HMW3 and its localization to the terminal portion of filamentous cell extensions. Most colloidal gold molecules were associated with the cell interior, but limited peripheral labeling of the terminal region was also observed. Postfixation antibody labeling of whole cells suggested limited exposure of HMW3 on the mycoplasma surface at the tip structure. However, prefixation antibody labeling failed to indicate surface exposure, raising some uncertainty regarding the relationship of HMW3 with the mycoplasma membrane.     

Electron microscopy of cells showing viral cytopathic effects in Papanicolaou smears

A technique is described whereby cells showing cytologic changes suggestive of virus infection by light microscopy can be processed further for examination in the electron microscope so that virus particles present in the cell can be visualized directly. We present the results of electron microscopy of over 100 Papanicolaou-stained smears processed this way. The morphologic changes in the cells and the ultrastructural appearances of the virus particles are demonstrated. This technique is particularly valuable for retrospective studies of mounted cytologic or histologic material. It has also proven to be a valuable research tool in the study of human polyomavirus and human wart virus infection.     

Immunocytochemical localization of histamine in enterochromaffin-like (ECL) cells in rat oxyntic mucosa: a transmission electron microscopy study using monoclonal antibodies and conventional glutaraldehyde-based fixation.

Histamine (HA), contained in the enterochromaffin-like (ECL) cells of the gastric mucosa in animals, plays an important role in gastric acid secretion, although methods for its exact morphological localization are still lacking. We used a pre-embedding indirect immunoperoxidase approach to define the fine structural localization of HA in rat oxyntic mucosa that was fixed with a glutaraldehyde-based fixative and HA monoclonal antibodies (MAbs AHA-1 and 2). Transmission electron microscopy showed that the peroxidase endproduct not only was concentrated in the cores of cytoplasmic granules but also was distributed to a high degree in the cytoplasm peripheral to the granules of the ECL cells. These results suggest that in ECL cells HA is enzymatically synthesized in the cytoplasm, then is transported and stored in the cores of the granules before its release from the basal lamina. The present HA immunoelectron microscopic method with MAbs would be applicable more generally to the ultrastructural identification of HA-containing cells.     

Postembedding immunocytochemical localization of paramyxovirus antigens by light and electron microscopy

A postembedding method is described to localize antigens specific for various paramyxoviruses in sections of cells and tissues that have been fixed and embedded in epoxy resins for conventional electron microscopy. Viral antigens were localized in CV-1 cell cultures infected with simian virus 5 (SV5), brains of suckling hamsters inoculated with either neuroadapted mumps virus or hamster-adapted measles virus, and brains of adult mice infected with Sendai (parainfluenza I) virus. Both 1-micrometer-thick and thin (gold) tissue sections were etched with alcoholic sodium hydroxide-solution and then treated following either the unlabeled antibody peroxidase-antiperoxidase or the biotinylated protein A:avidin peroxidase procedure. Primary reagents included immunoglobulin isolated from hyperimmune rabbit sera with specificity to the major viral components of SV5 or SV5 hemagglutinin-neuraminidase, to whole mumps virus or mumps virus nucleocapsids, and to whole Sendai virus. Crude rabbit anti-Sendai virus antiserum and whole human subacute sclerosing panencephalitis (SSPE) sera were used in parallel. The results indicate that tissues processed for conventional evaluation by electron microscopy may be suitable, within limits, for postembedding immunocytochemical staining of paramyxovirus antigens.     

Characterization of a new neurosecretory cell type containing gastrin-cholecystokinin-like peptide in the locust pars intercerebralis: ultrastructural and immunocytochemical studies,in situ and in vitro

A new neurosecretory cell type of the locust pars intercerebralis, immunolabelled with an antiserum against a vertebrate peptide related to gastrin-cholecystokinin (CCK-8(s)), was characterized both in situ and in primary cell cultures. Semithin sections of pars intercerebralis were first immunostained in order to identify neurosecretory cells containing CCK-like material and then examined by electron microscopy. The neurosecretory cells containing CCK-like material were paraldehyde fuchsin negative and were unequivocally identified in ultrathin sections adjacent to immunostained semithin sections. They exhibited neurosecretory vesicles of variable electron density, ranging in diameter from 150 to 250 nm. Immunogold labelled ultrathin sections adjacent to unlabelled ultrathin sections allowed for the unambiguous localization of CCK-like immunoreactive material over the neurosecretory vesicles of the cells containing CCK-like material. Immunoreactivity towards CCK-8(s)-like peptide could also be detected in pars intercerebralis neurosecretory neurons grown in vitro. The CCK-like positive neurons showed a multipolar morphology with fine processes radiating from the cell body. The positive cells had the same ultrastructural characteristics as the in situ CCK-like neurons. The pattern of neurite outgrowth on reactive CCK-like neurosecretory cells in vitro and the neuroanatomical pathway of the CCK-like immunoreactive neurosecretory cells in situ could be correlated. On the basis of their number, size and localization in the locust pars intercerebralis, it is possible that the CCK-like neurosecretory cells correspond to neurosecretory cell type C, which has not, to date, been identified at the ultrastructural level.     

Identification of enterochromaffin cells in adjacent Epon-embedded sections at light and electron microscopic levels

Summary Methods for light and electron microscopic comparison of individual argentaffin and argyrophil enterochromaffin cells (EC) in the sheep duodenal mucosa are described. These silver procedures were applied for light microscopy to Epon-embedded sections. The adjacent sections were examined with the electron microscope. The most specific characteristics of the argentaffin and argyrophil EC in electron microscopy are highly osmiophilic cytoplasmic granules. In one cell type these granules are smaller and more roundish than in the another type. These two cell types are stainable both by the argentaffin and argyrophil reactions. No essential difference can be observed in the localization of these elements. It is suggested that both cell types belong to the enterochromaffin system. Both silver methods are also suitable for the light microscopic identification of other intestinal structures in sections adjacent to that sectioned for electron microscopy.This work was supported by a grant from the Yrjö Jahnsson Foundation, Helsinki, Finland.The electron microscopic observations were carried out in the Electron Microscope Laboratory, University of Helsinki.