In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.     

In situ hybridization of iodinated ribosomal RNA to human chromosomes

Iodinated ribosomal RNA was hybridized to human metaphase chromosomes in a test of the effectiveness of iodinated products for in situ hybridization studies in a diploid system. The results indicate that ¹²⁵I is a feasible alternative as a source of radioactivity for autoradiographic mapping studies.     

In situ hybridization at the electron microscope level: localization of transcripts on ultrathin sections of Lowicryl K4M-embedded tissue using biotinylated probes and protein A-gold complexes

A technique has been developed for localizing hybrids formed in situ on semi-thin and ultrathin sections of Lowicryl K4M-embedded tissue. Biotinylated dUTP (Bio-11-dUTP and/or Bio-16-dUTP) was incorporated into mitochondrial rDNA and small nuclear U1 probes by nick-translation. The probes were hybridized to sections of Drosophila ovaries and subsequently detected with an anti-biotin antibody and protein A-gold complex. On semi-thin sections, probe detection was achieved by amplification steps with anti-protein A antibody and protein A-gold with subsequent silver enhancement. At the electron microscope level, specific labeling was obtained over structures known to be the site of expression of the appropriate genes (i.e., either over mitochondria or over nuclei). The labeling pattern at the light microscope level (semi-thin sections) was consistent with that obtained at the electron microscope level. The described nonradioactive procedures for hybrid detection on Lowicryl K4M-embedded tissue sections offer several advantages: rapid signal detection: superior morphological preservation and spatial resolution; and signal-to-noise ratios equivalent to radiolabeling.     

Anti-bromodeoxyuridine monoclonal antibody: an alternative tool for the identification of replicated DNA at the electron microscope level

A new method for identifying the replicated DNA at the electron microscope level is described. Cells were first exposed in vitro to 5-bromodeoxyuridine (BUdR) in conjunction with 5-fluorodeoxyuridine (FUdR) and BUdR incorporated into DNA was then detected on Lowicryl-embedded sections by immunogold technique using a monoclonal anti-BUdR antibody. After using this method, chromatin and chromosomes are strongly labelled.     

Localization of ribosomal cistrons at the ultrastructural level by in situ hybridization technique

In situ hybridization with 3H 28 S ribosomal RNA at the ultrastructural level shows labelling exclusively over the nucleolus and specifically over the dense nucleolar component (DNC). These findings clearly reveal that the ribosomal cistrons are located in the DNC of the nucleolus.     

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.     

A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochrome-labelled RNA

A new method has been developed for the detection of in situ hybridization by fluorescence microscopy. It is based on the covalent binding of commercially available fluorochromes to the 3′-terminus of RNA.     

Development of a procedure for autoradiographic localization of carbonic anhydrase at the electron microscope level

A method for preparing tissue suitable for electron microscope autoradiographic localization of carbonic anhydrase is described. Radioactivity is in the form of 3H-acetazolamide, a specific inhibitor of carbonic anhydrase. Tissues fixed in glutaraldehyde, dehydrated in ethylene glycol followed by cellosolve as a transition fluid, and embedded in epoxy resin, were found to retain most (74%) of the label. Electron micrographs of avian gastric mucosa prepared in this manner are shown. Other methods of preparation were explored and resulted in considerable losses of label or in inadequately preserved tissue. Light microscope autoradiographic localization of gastric mucosa, shell gland, chorioallantoic membrane, and skeletal muscle compare well with previous localizations.     

More precise localization of the human factor IX gene by in situ hybridization

The location of the human antihemophilic Factor IX has been more specifically assigned from the region Xq27----qter to Xq26----q27 by quantitative in situ hybridization. The present study utilized a complex hybridization probe and prephotographed G-banded human chromosomes to improve analytical sensitivity and accuracy.     

Chromosomal in situ hybridization with double-labeled DNA: signal amplification at the probe level.

A double-labeling approach was applied to nonisotopic in situ hybridization with individual cosmid and plasmid clones, using digoxigenin or biotin as label and a combination of two separate enzymatic labeling methods. Probe labeling was achieved by nick translation, followed by tailing of the probe by terminal deoxynucleotidyl transferase. The double-labeling method, in conjunction with an improved detection protocol, provides for a higher signal intensity than that obtainable with single-labeled probes.     

Immunohistochemical localization of c-mos at the light and electron microscope level in non-small cell lung carcinomas

C-mos is a cytoplasmic upstream activator of the mitogen-activating protein kinase pathway with serine-threonine kinase activity. It plays a well established and vital role in oocyte maturation by participating in metaphase II arrest and meiotic asymmetric division, but little is known about its function in somatic cells. Recently, we observed overexpressed c-mos in a portion of non-small cell lung carcinomas (NSCLCS). In particular, c-mos immunoreactivity was detected in tumor cell nuclei in addition to its expected cytoplasmic localization, and c-mos overexpression was associated with chromosomal instability among other findings. To verify our earlier observations and to clarify further the role of c-mos in NSCLCS, we examined its distribution by both light and electron microscopy. We detected c-mos in the cytoplasm and/or nucleus of a portion of tumor cells and fibroblasts. In particular, granular immunoreactivity was observed in the cytoplasm closely associated with the rough endoplasmic reticulum. Nuclear staining was confirmed and was often found near the nuclear membrane, as well as in some large multilobular, possibly aneuploid, nuclei. C-mos positivity was also found in the nuclei of tumor cells undergoing apoptosis. Furthermore, c-mos was detected in areas with diminished vascularization. It should be noted that nuclear staining was found at the ultrastructural level more extensively than at the light microscope study. This suggests a masking effect by the hematoxylin nuclear counterstain.     

Preliminary karyotype and chromosomal localization of ribosomal DNA sites in white spruce using fluorescence in situ hybridization.

We have localized the major ribosomal DNA (rDNA) loci on metaphase chromosomes and in interphase nuclei of white spruce (2n = 24) by fluorescence in situ hybridization. Hybridization sites of the biotin-labelled rDNA probe were detected using antibody-fluorochrome conjugates and a confocal laser scanning microscope. White spruce has at least 12, and possibly as many as 14, rDNA sites, 1 site present on each of seven separate chromosome pairs. This is one of the highest numbers of rDNA loci yet reported among plant species. The position of the rDNA loci together with secondary constriction patterns permit, for the first time, all homologous pairs of white spruce chromosomes to be distinguished. We discuss the application of molecular cytogenetics in studies relating to the organization and evolution of DNA sequences within conifer genomes.     

In situ hybridization of ribosomal DNA labelled with 125iodine to metaphase and lampbrush chromosomes from newts

Methods are described for in situ hybridization of ribosomal DNA from Xenopus laevis, labelled in vitro with ¹²⁵iodine, to mitotic and lampbrush chromosomes from Triturus cristatus carnifex. The hybridization reaction was carried out in a mixture containing 50% formamide, 4 x SSC, 0.1 M KI, at 37° C, or in 2 x SSC, 0.1 M KI at 65° C. Autoradiographs of mitotic metaphases from 2 males showed labelling over the middle of the short arm of one chromosome IX in each metaphase. In some cases, a region near the end of a longer chromosome was also labelled. In lampbrush preparations, labelling was confined to a region identified as about 53 units, near the middle of the short arm of both halves of bivalent IX. The usefulness of the technique and the significance of the labelling of only 1 of the 2 chromosomes IX in mitotic preparations are discussed.