Ultrastructural detection of ribosomal RNA by in situ hybridization using a biotinylated probe on ultrathin sections of cultured animal cells

A genomic probe containing ribosomal sequences and labelled with biotin was used to hybridize rRNA molecules in ultrathin sections of animal cells embedded in Lowicryl K4M. After detection with streptavidin conjugated with 10 nm gold particles, ribosomal target sequences were localized preferentially in the dense fibrillar component of the nucleolus and in the polyribosome structures of the cytoplasm. The method presently described offers the possibility to detect rapidly and precisely ribosomal gene expression at the ultrastructural level, particularly under different physiological and pathological conditions.     

In situ ultrastructural localization of sugar-binding sites in lizard granulosa cell nuclei

Mannose-binding sites were detected at the ultrastructural level in nuclei of lizard granulosa cells in situ by means of mannosylated ferritin. When ultrathin sections of material embedded in glycol methacrylate were incubated with mannosylated ferritin, a strong labelling was observed over nucleoli, chromatin and the external leaflet of the nuclear envelope, but no labelling was detected in the perinuclear space except for nuclear pores. This labelling was clearly inhibited when sections were incubated in a solution containing both mannosylated ferritin and a sugar-related neoglycoprotein (mannosylated serum albumin). These ultrastructural data supporting the existence of nuclear lectin-like components in reptilian cells are in agreement with our previous findings about such components in nuclei isolated from mammalian cells. Owing to the unique organization of the granular component of the nucleoli in specialized granulosa cells, we are able to show that some of the mannose-binding sites are associated with ribosomal precursors.     

Ultrastructural non-radioactive in situ hybridization of GH mRNA in rat pituitary gland: pre-embedding vs ultra-thin frozen sections vs post-embedding.

In situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultra-thin frozen sections, or on ultra-thin sections of tissues embedded in hydrophilic resin such as Lowicryl. With the purpose of comparing the sensitivity, resolution, and ultrastructural preservation of these three methods, we examined the expression of the growth hormone (GH) gene in anterior pituitary cells by in situ hybridization at the ultrastructural level, using a synthetic oligonucleotide complementary to the codons of the mRNA from Gln 45 to Ser 54 labeled at the 3' end of biotin-21dUTP. All these methods gave similar results: mRNA was located on the lamellar endoplasmic reticulum of somatotrophs. The pre-embedding method gave the best ultrastructural preservation, with low resolution with the enzymatic detection system and an intermediate sensitivity. A probe concentration of 10 pmol/ml was sufficient to obtain a signal. With this method gold particles could not be used without pre-treatment. The frozen section method gave the best sensitivity (a signal was observed with 4 pmol/ml of probe) but the lowest ultrastructural preservation. On ultra-thin Lowicryl sections, resolution was as high as with the frozen-section method, ultrastructural conservation was intermediate, and sensitivity was low. These results indicate that the last method seems to be a good compromise between sensitivity and ultrastructural preservation.     

Activation of guinea pig peritoneal macrophages by ribosomal extract of Salmonella typhi strain. Electron microscopy studies

Normal macrophages and Salmonella typhi ribosomal antigen-activated (immune) ones were studied by electron microscopy. Examination of fine sections from samples incubated between 30 and 120 minutes at 37 degrees C, emphasized phagocytic ability of activated macrophages (29-69% activated macrophages phagocytized 3.7-5.6 bacteria/cell, in contrast to 23-43% with 2.6-4.4 bacteria/phagocytized by non-immune cells). The ultrastructural studies of activated macrophages, performed by electron microscopy, showed the following aspects: a) macrophages with pseudopodiform prolongations, presenting the tendency of bacterial sequestration: b) phagosomes with ingested virulent germs; c). bacteria presenting various degrees of wall and cytoplasm alterations; d). mitochondria with multiple cristae; e). cells with well developed Golgi apparatus and the presence of lysosomes in great numbers; f). existence of a space between the phagosome membrane and the bacterial wall. Experimental results demonstrated: I. an increased phagocytic activity of Salmonella typhi ribosomal antigen-activated macrophages and II. activation expressed as ultrastructural modifications at the level of immune macrophages and also of the bacteria phagocytized by them.     

An ultrastructural study of bruising in stored potato (Solarium tuberosum L.) tubers

Potato tubers of the cultivar Pentland Dell were investigated for ultrastructural changes following impact. Studies were carried out on tubers after 12 and 24 wk of storage using a falling bolt delivering 0.7 J of energy. Ultra-thin sections were examined by light and transmission electron microscopy. Tubers stored for 12 wk exhibited no visual bruise formation, whereas visible bruising resulted following impact of tubers stored for 24 wk. An ultrastructural sequence of events during bruising was established as (1) a collapse of intracellular compartmentation, (2) increased ribosomal and mitochondrial abundance within the cytoplasm, (3) increased density of cytoplasm adjacent to the cell wall and surrounding amyloplasts, and (4) the development of melanin in bruised cells. These observations are discussed in relation to tuber turgor and physiology during storage.     

Detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp by in situ hybridization at the electron microscope level

A post-embedding in situ hybridization procedure was developed to detect hepatopancreatic parvovirus (HPV) of penaeid shrimp at the ultrastructural level. The procedure was optimized using sections of resin-embedded hepatopancreas from HPV-infected juvenile Penaeus monodon and postlarval P. chinensis. The hepatopancreata were fixed using various fixatives, dehydrated, and embedded in the hydrophilic resin Unicryl. A 592 bp HPV-specific DNA probe, labeled with DIG-11-dUTP, was tested both on semi-thin and ultra-thin sections and examined by light and electron microscopy, respectively. Hybridized probe was detected by means of an anti-DIG antibody conjugated to 10 nm gold particles and subsequent silver enhancement. Hybridization signal intensities were similar with all fixatives tested, but ultrastructure was best preserved with either 2 or 6% glutaraldehyde. Post-fixation with 1% osmium tetroxide improved ultrastructure but markedly decreased hybridization signal and induced non-specific deposition of gold and silver. Under optimized conditions, this technique was used to successfully follow the development of HPV from absorption and transport through the cytoplansm to nuclear penetration, replication and release by cytolysis. The probe signal was consistently observed among necrotic cell debris within the lumen of hepatopancreatic tubules, within the microvillous border of tubule epithelial cells, within the cytoplasm, and within diagnostic HPV intranuclear inclusion bodies. The nucleolus and karyoplasm of patently infected cells (i.e., showing HPV intranuclear inclusion bodies) were almost devoid of signal. Electron-lucent structures, known as intranuclear bodies, commonly found within the virogenic stroma, showed only weak labeling. This is the first use of in situ hybridization to detect HPV nucleic acids with the electron microscope. The technique should be useful for studying the pathogenesis of HPV.     

In situ localization of transketolase activity in epithelial cells of different rat tissues and subcellularly in liver parenchymal cells.

Metabolic mapping of enzyme activities (enzyme histochemistry) is an important tool to understand (patho)physiological functions of enzymes. A new enzyme histochemical method has been developed to detect transketolase activity in situ in various rat tissues and its ultrastructural localization in individual cells. In situ detection of transketolase is important because this multifunctional enzyme has been related with diseases such as cancer, diabetes, Alzheimer's disease, and Wernicke-Korsakoff's syndrome. The proposed method is based on the tetrazolium salt method applied to unfixed cryostat sections in the presence of polyvinyl alcohol. The method appeared to be specific for transketolase activity when the proper control reaction is performed and showed a linear increase of the amount of final reaction product with incubation time. Transketolase activity was studied in liver, small intestine, trachea, tongue, kidney, adrenal gland, and eye. Activity was found in liver parenchyma, epithelium of small intestine, trachea, tongue, proximal tubules of kidney and cornea, and ganglion cells in medulla of adrenal gland. To demonstrate transketolase activity ultrastructurally in liver parenchymal cells, the cupper iron method was used. It was shown that transketolase activity was present in peroxisomes and at membranes of granular endoplasmic reticulum. This ultrastructural localization is similar to that of glucose-6-phosphate dehydrogenase activity, suggesting activity of the pentose phosphate pathway at these sites. It is concluded that the method developed for in situ localization of transketolase activity for light and electron microscopy is specific and allows further investigation of the role of transketolase in (proliferation of) cancer cells and other pathophysiological processes.     

A specific ultrastructural method to reveal DNA: the NAMA-Ur.

We have developed a new, simple, and reproducible cytochemical method to specifically stain DNA at the electron microscopic level: the NAMA-Ur. It is based on the extraction of RNA and phosphate groups from phosphoproteins by a weak alkali hydrolysis (NA) which does not affect DNA, followed by blockage of the amino and carboxyl groups by methylation and acetylation (MA). Finally, sections are stained by uranyl (Ur), which can bind only to DNA. The efficiency of the pre-treatment (NA and MA) was demonstrated by X-ray microanalysis at the transmission electron microscopic level. The NAMA-Ur method has been successfully performed en bloc and on Lowicryl sections in mammalian and plant cells. A specific contrast is observed in the DNA-containing structures after this method, whose sensitivity allows visualization of electron-dense chromatin fibers of 10-12 nm composed of 3-nm DNA fibrils. This staining method has been combined with anti-DNA antibodies, providing complementary information to detect DNA in situ. We propose the NAMA-Ur as an easy method to investigate the chromatin organization in situ at the ultrastructural level.     

Electron microscope analysis of tissue components identified and located by computer-assisted 3-D reconstructions: ultrastructural segmentation of the developing human proximal tubule

A method is described for ultrastructural analysis of renal tubules after precise identification of tubule segments by computerized 3-D reconstruction at the light microscope level. Semithin serial sections were cut of entire nephrons and 3-D coordinate information was obtained by digitization of tubule cross sections in the semithin sections. With the aid of the computer the tubule axis was traced from one section to the other. Precise lengths and positions of the tubules in three dimensions were calculated and stereoscopic images generated. The method was used to analyze the 3-D structure of developing human nephrons, and the ultrastructural development of the proximal tubule. Ultrastructural segmentation of the proximal tubule was demonstrated in the human fetal nephron in developmental stage IV.     

Highly sensitive immunodetection of DNA on sections with exogenous terminal deoxynucleotidyl transferase and non-isotopic nucleotide analogues.

A new method is described for locating DNA on ultra-thin sections. Sections of aldehyde-fixed, plastic-embedded cells were incubated in a medium containing terminal deoxynucleotidyl transferase (TdT) and various non-isotopic nucleotide analogues. The labeled nucleotides bound to the surface of ultra-thin sections were then visualized by an indirect immunogold labeling technique. The resulting labeling pattern was strongly dependent on the divalent cation used in the TdT medium. The method revealed with great precision the specific DNA-containing structures within Ehrlich tumor cells, even where DNA was present in very low amounts. The method is compatible with all usual fixation and embedding procedures and can be combined with cytochemical methods. The in situ TdT method provides a very useful tool for pinpointing the precise location of DNA within biological material at the ultrastructural level.     

Ultrastructural cytochemical, immunocytochemical and in situ hybridization methods with polyuridine probes detect mRNA in human mast cell granules

Mature human mast cells are classical secretory cells that are filled with secretory-storage granules but are poorly endowed with visible free or membrane-bound cytoplasmic ribosomes. We recently reported close associations of ribosomes and various components essential to RNA metabolism in and close to human mast cell granules using multiple ultrastructural imaging methods. In view of these findings and an increased awareness of RNA sorting and localization to specific subcellular sites and organelles, we used human mast cells purified from non-tumour portions of lung samples resected at surgery for carcinoma and ultrastructural methods to investigate this further. Poly(U) probes were used to detect direct en grid binding, and radiolabelled as well as non-radiolabelled poly(U) probes were used in in situ hybridization protocols to detect poly(A)-positive pre-mRNA and mRNA in nuclear, cytoplasmic and granular compartments of mature human mast cells. Negative controls verified specificity of label; expected nuclear and cytoplasmic locations of poly(A)-positive RNA served as positive controls for each sample. These findings lend support to the hypothesis that site-specific synthesis in secretory-storage granules may occur in secretory cells.     

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.     

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.     

A TECHNIQUE FOR ULTRACRYOTOMY OF CELL SUSPENSIONS AND TISSUES

Ultracryotomy of fixed tissue has been investigated for a number of years but, so far, success has been limited for several reasons. The simple technique herein reported allows the ultracryotomy not only of a variety of tissues but also of single cells in suspension, with a preservation and visualization of ultrastructural detail at least equivalent to that obtained with conventional embedding procedures. In this technique, sucrose is infused into glutaraldehyde-fixed tissue pieces before freezing for the purpose of controlling the sectioning consistency. By choosing the proper combinations of sucrose concentration and sectioning temperature, a wide variety of tissues can be smoothly sectioned. Isolated cells, suspended in a sucrose solution, are sectioned by sectioning the frozen droplet of the suspension. A small liquid droplet of a saturated or near-saturated sucrose solution, suspended on the tip of an eyelash probe, is used to transfer frozen sections from the knife edge onto a grid substrate or a water surface. Upon melting of the sections on the surface of the sucrose droplet, they are spread flat and smooth due to surface tension. When the section of a suspension of single cells melts, individual sections of cells remain confined to the small area of the droplet surface. These devices make it possible to cut wide dry sections, and to avoid flotation on dimethyl sulfoxide solutions. With appropriate staining procedures, well-preserved ultrastructural detail can be observed. The technique is illustrated with a number of tissue preparations and with suspensions of erythrocytes and bacterial cells.     

CD56(NCAM) antigen in glandular epithelium of human thyroid: light microscopic and ultrastructural study

CD56 antigen, an isoform of the neural cell adhesion molecule (NCAM) was previously found by us in human thyroid by APAAP immunohistochemistry in light microscopy on frozen tissue sections. In the current study, it was attempted to trace the antigen in question using another light microscopic immunohistochemical procedure and to validate the results at the ultrastructural level. For light microscopy, cryostat sections of 12 surgical samples of human thyroid were subjected to ABC (preformed avidin-biotin-peroxidase complex) method. For immunoelectron microscopy, immunoperoxidase reaction was carried out on prefixed, small thyroid tissue blocks. Following preliminary inspection of semithin sections, ultrathin sections were examined in the transmission electron microscope. ABC reaction revealed distinct specific CD56 staining of thyrocyte cell membranes. The staining was weak or absent in thyroid papillary carcinoma cells. The results were confirmed in semithin sections by indirect immunoperoxidase. The latter reaction in ultrathin sections at the ultrastructural level has shown that specific reaction product was confined to free and lateral surfaces of thyroid follicular cells. Endothelial cell membranes of thyroid capillary vessels were totally devoid of the reaction product. The reaction was weakly positive in thyroid follicular and papilllary carcinomas but absent from medullary carcinoma.     

Gold and Female Reproductive Organs: an Ultrastructural Study

Gold, a heavy yellow-colored metal, is usually found in nature as a metallic element or as salts. This noble metal historically had a reputation as an anti-inflammatory medicine for rheumatoid arthritis, a nervine, and a remedy for nervous disorders, as well as a potential anticancer agent. It has also been used as component in dental restorations and in implant materials. The present study was undertaken to point out histological and ultrastructural effects of gold, administered by intraperitoneal route, in pregnant female reproductive organs (ovary and uterus), in order to clarify its side effects on the reproductive function. Using the transmission electron microscopy (TEM), the ultrastructural investigations of both ultrathin ovarian and uterine sections of treated pregnant rats revealed the existence of numerous heterogeneous clusters with very electron-dense inclusions characterized by various aspects in the lysosomes of granulosa, theca interna cells, and theca externa cells. Degeneration of these tissues, like cell vacuolization, marked expansion of the endoplasmic reticulum, mitochondrial alterations, and necrotic foci, were also highlighted. Moreover, huge phagolysosomes and high numbers of eosinophils as signs of inflammation were also identified especially in endometrial and myometrial cells of gold-treated rats. The ultrastructural investigations of reproductive organ sections of control pregnant rats showed a normal ultrastructural aspect and no loaded lysosomes. These results speculated the toxicity of gold at the used dose. The observed signs of toxicity allowed concluding that the important role of lysosome in the sequestration of this element under an insoluble form in all categories of cells in the studied tissues does not seem to be efficient.     

Development of the endocrine pancreas during larval phases of Rana temporaria

Summary The pancreatic endocrine component was studied at different stages of development in the tadpoles of Rana temporaria. The material was embedded in Epon, and serial semithin and thin sections were made in order to correlate ultrastructural features and tinctorial traits of the endocrine cells. Serial semithin sections were also stained with the peroxidase-antiperoxidase immunocytochemical method and with silver impregnations for argyrophilia and argentaffinity. In early larvae (legless tadpoles), A and B cells are present. Both can be found within ducts and exocrine tissue or, more frequently, in cellular clusters among the ducts and acini. These primitive islets are solid structures, surrounded but not penetrated by capillaries. Mitoses were observed in A and B cells. In the following phase (tadpoles with hindlegs), D and pancreatic polypeptide-immunoreactive cells are also present, as well as numerous endocrine cells scattered among exocrine tissue. There is also a change in the vascular-insular pattern: capillaries not only surround but also penetrate the endocrine group. The structure of the endocrine pancreas in older tadpoles is similar. Tinctorial traits and ultrastructural features of endocrine cells are described, and the origin of primitive islets is discussed.     

Polymyxin B binding sites in Escherichia coli as revealed by polymyxin B-gold labeling

A complex of polymyxin B, bovine serum albumin, and colloidal gold was prepared and used for the ultrastructural localization of polymyxin B binding sites on thin sections of Epon-embedded Escherichia coli cells. Gold particles were found on the outer membrane of E. coli, which is consistent with reported biochemical findings. We concluded that gold labeling with polymyxin B is useful in localizing the binding sites of polymyxin.     

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.