Use of a biotinylated probe and in situ hybridization for light and electron microscopic localization of Po mRNA in myelin-forming Schwann cells

A biotinylated Po glycoprotein cDNA was hybridized in situ to aldehyde-fixed vibratome sections and to aldehyde-fixed thin sections of Lowicryl-embedded trigeminal ganglia of 15 day old rats. Alkaline phosphatase and peroxidase detectors were used for light microscopic (LM) studies and peroxidase or colloidal gold were employed for electron microscopic (EM) detection. In both LM and EM sections, probe was found in cytoplasmic areas of myelin-forming Schwann cells that were enriched in granular endoplasmic reticulum, demonstrating that these regions contain Po mRNA. Interestingly, Po mRNA tended to cluster in regions close to the developing myelin sheath. Relatively simple methods are here described for EM detection of mRNA with reasonable tissue preservation and high resolution. These methods may be useful for developmental and disease-related studies of specific mRNAs in mammalian tissues.     

Localization of mRNA for pea legumin:In situ hybridization using a biotinylated cDNA probe

Summary In situ hybridization has been used to locate mRNA, for the storage protein legumin, in cotyledon storage parenchyma tissue of developing pea (Pisum sativum L.) seeds. The mRNA was hybridized with a biotinylated probe of cDNA in pBR 322 and subsequently located by avidin conjugates. Avidin-rhodamine was used for fluorescence microscopy localization at a tissue/cellular level and avidin-peroxidase (with DAB) and avidin-ferritin compared for localization at an ultrastructural level. Specific fluorescence associated with avidin-rhodamine was distributed unevenly throughout the cytosol but the cell walls, starch grains, vacuoles and protein deposits were unstained. The sizes and distribution of the regions of higher labeling within the cytosol suggest an association with elements of the endomembrane system. Following DAB reaction of the specifically localized avidin-peroxidase most, although not all, stain product was associated with the endoplasmic reticulum. The ER-associated reaction product was also accumulated within the ER lumen.Avidin-ferritin was also localized both in the cytosol and in association with the endoplasmic reticulum, although was less readily visualized in cells with a conventional ultrastructural appearance.Localization of avidin-ferritin was more readily visualized in cells which had undergone some limited structural damage during specimen preparation. In such cases ferritin was also shown to be specifically associated with the transition vesicles and trans-face peripheral vesicles of some dictyosomes.     

Detection of proopiomelanocortin mRNA by in situ hybridization, using a biotinylated oligodeoxynucleotide probe and avidin-alkaline phosphatase histochemistry

A synthetic 24-mer oligodeoxynucleotide complementary to the region of proopiomelanocortin (POMC) mRNA that codes for the MSH core sequence (alpha MSH/ACTH[4-11]), was synthesized and labelled in the 3'-end by use of terminal transferase. Probes tailed with either [3H]- or biotin-labelled nucleotides could be used for in situ hybridization studies. Biotinylated probes, hybridized to mouse and rat pituitary sections, were detected by avidin-alkaline phosphatase or streptavidin-alkaline phosphatase procedures and development in 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium (NBT). Proteinase K pretreatment of sections produced a drastic enhancement of the signal obtained, particularly in strongly fixed, paraffin-embedded material. The non-radioactive in situ hybridization technique compared favourably to radioactive in situ hybridization in terms of rapidity and precision of the localization. Controls involved deletion of the probe to prove that other components of the reaction sequence did not yield stain, digestion with RNase to prove that tissue RNA was necessary to bind the probe, prehybridization (blocking) with unlabelled probe to prove that the biotinylated probe reacted with its anti-sense region and not its tail and Northern blotting to show that the probe reacted with only one species of pituitary RNA, having the size of mouse pituitary POMC mRNA. In addition, adrenalectomy, known to increase anterior lobe POMC levels, resulted in both increased numbers and increased intensity of positive corticotroph-like cells. Synthetic oligodeoxynucleotides labelled with biotin appear to constitute attractive reagents for in situ hybridization studies when supported by appropriate control procedures.     

Detection of proopiomelanocortin mRNA by in situ hybridization,using a biotinylated oligodeoxynucleotide probe and avidin-alkaline phosphatase histochemistry

Summary A synthetic 24-mer oligodeoxynucleotide complementary to the region of proopiomelanocortin (POMC) mRNA that codes for the MSH core sequence (MSH/ACTH[4-11]), ws synthesized and labelled in the 3-end by use of terminal transferase. Probes tailed with either [³H]- or biotin-labelled nucleotides could be used for in situ hydridization studies. Biotinylated probes, hybridized to mouse and rat pituitary sections, were detected by avidinalkaline phosphatase or streptavidin-alkaline phosphatase procedures and development in 5-brome-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium (NBT). Proteinase K pretreatment of sections produced a drastic enhancement of the signal obtained, particularly in strongly fixed, paraffin-embedded material. The non-radioactive in situ hybridization technique compared favourably to radioactive in situ hybridization in terms of rapidity and precision of the localization. Controls involved deletion of the probe to prove that other components of the reaction sequence did not yield stain, digestion with RNase to prove that tissue RNA was necessary to bind the probe, prehybridazation (blocking) with unlabelled probe to prove that the biotinylated probe reacted with its anti-sense region and not its tail and Northern blotting to show that the probe reacted with only one species of pituitary RNA, having the size of mouse pituitary POMC mRNA. In addition, adrenalectomy, known to increase anterior lobe POMC, levels, resulted in both increased numbers and increased intensity of positive corticotroph-like cells. Synthetic oligodeoxynucleotides labelled with biotin appear to constitute attractive reagents for in situ hybridization studies when supported by appropriate control procedures.     

Tissue-plasminogen activator RNA detected in megakaryocytes by in situ hybridization and biotinylated probe

Summary Tissue-plasminogen activator (t-PA) has been identified in human megakaryocytes isolated from human rib fragments or from culture cells. The presence of t-PA antigen in the endoplasmic reticulum of mature cells was demonstrated by ultrastructural immunochemistry. These results suggest that t-PA can be synthesized by megakaryocytes as well as by vascular endothelial cells. In order to confirm this possibility, in situ hybridization using antisense RNA biotinylated probe was used in megakaryocyte culture at different steps of maturation. The presence of t-PA-RNA was clearly demonstrated at an early step of differentiation. Biotin staining was enhanced in mature cells and this was correlated with the detection of t-PA antigen by immunocytochemistry.     

Tissue-plasminogen activator RNA detected in megakaryocytes by in situ hybridization and biotinylated probe

Tissue-plasminogen activator (t-PA) has been identified in human megakaryocytes isolated from human rib fragments or from culture cells. The presence of t-PA antigen in the endoplasmic reticulum of mature cells was demonstrated by ultrastructural immunochemistry. These results suggest that t-PA can be synthesized by megakaryocytes as well as by vascular endothelial cells. In order to confirm this possibility, in situ hybridization using antisense RNA biotinylated probe was used in megakaryocyte culture at different steps of maturation. The presence of t-PA-RNA was clearly demonstrated at an early step of differentiation. Biotin staining was enhanced in mature cells and this was correlated with the detection of t-PA antigen by immunocytochemistry.     

HSV hepatitis in the mouse: a light and electron microscopic study with immunohistology and in situ hybridization

In order to characterize better the morphology and immune response in acute necrotizing HSV infection, murine HSV hepatitis was examined. BALB/c mice were inoculated intraperitoneally with 10(6) plaque-forming units (PFU) of HSV-1 (Lenette) and HSV-2 (D316). In both groups half the animals were pretreated with silica particles to block macrophage function. Up to 6 days after infection four mice from each group were sacrificed at daily intervals and the livers were examined by light and electron microscopy, immunohistology, in situ hybridization, combined immunohistology/in situ hybridization and titration of viral PFU. HSV-2 infected mice developed severe necrotizing hepatitis with persistence of HSV in the liver tissue until the end of the study. HSV-1 infected mice rapidly eliminated the virus and revealed only small necrotic foci. Early phase alterations and necrotic phase lesions were distinguished and characterized and morphologic evidence of a direct cytopathic effect of HSV was detected. A specific immune reaction in late stages appeared to be mediated by T4-positive T-lymphocytes. In situ hybridization and immunohistochemistry showed a close correlation with virus titration and were valuable in characterizing early phases and in the assessment of prognosis and differential diagnosis.     

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.     

Immunohistochemical localization of albumin and in situ hybridization of albumin mRNA

Hepatocytes actively involved in albumin synthesis were identified by immunohistochemical method. In sections of perioidate-lysine-2 per cent (w/v) paraformaldehyde fixed normal rat liver, albumin was detected in all hepatocytes. At the ultrastructural level, albumin was localized in the rough endoplasmic reticulum and in Golgi complexes located near the nucleus in only a small subpopulation of hepatocytes, while all other hepatocytes contained albumin only in Golgi complexes located near the bile canaliculi. Stimulation of albumin synthesis by puromycin aminonucleoside-induced nephrosis resulted in an altered intracellular distribution of albumin at the light microscopic level. When examined at the ultrastructural level, albumin was localized in the rough endoplasmic reticulum as well as in Golgi complexes located near the nucleus in nearly all these hepatocytes. Hepatocytes with the potential to synthesize albumin were identified by in situ hybridization of albumin mRNA. In sections of 0.1 per cent (v/v) glutaraldehyde perfusion fixed normal rat liver, albumin mRNA was detected in the cytoplasm of only a few hepatocytes scattered throughout the lobule. Following stimulation of albumin synthesis by the induction of nephrosis, albumin mRNA was detected in the cytoplasm of the hepatocytes. The source of albumin in those hepatocytes which lacked albumin mRNA was identified in analbuminemic rats injected with rat albumin. At 6 h post injection, the light microscopic distribution of albumin in the liver of these animals was virtually indistinguishable from that in normal rat liver. At the ultrastructural level, injected albumin was localized in lysosomes and in Golgi complexes located near the bile canaliculi.     

Chromosomal localization of genes by scanning electron microscopy using in situ hybridization with biotinylated probes: Y chromosome repetitive sequences

Summary The feasibility of using scanning electron microscopy (SEM) to identify the position of specific DNA sequences was examined using a Y chromosome specific probe (pHY2.1). Tests were carried out on chromosome spreads hybridizedin situ with biotinylated pHY2.1. Chromosomal sites of hybridization of the probe were localized by an indirect immunohistochemical procedure which resulted in a gold product which could be amplified by silver precipitation. In the SEM, the specific location of the probe was easily identified due to the enhanced signal produced by the gold—silver complex. The probe was localized both on the long arm of the Y chromosome and within interphase nuclei. It was found that SEM was more sensitive than light microscopy since the probe could be identified without silver amplification. With refinements to the technique, SEM could provide a useful method for high resolution localizing of unique DNA sequences (i.e. single copy genes).     

Evaluation of pepsin treatment for electron microscopic RNA in situ hybridization on ultra-thin cryosections of cultured cells

The in situ hybridization (ISH) technique, as applied to electron microscopic detection of RNAs, was evaluated for ultra-thin cryosections of cultured rat fibroblasts (rat 9G). Experimental variables to balance penetration of detection reagents and preservation of ultrastructural morphology included various strengths of aldehyde fixation and pepsin treatment. We performed ISH for 28S ribosomal RNA (rRNA) followed by ultra-small colloidal gold immunocytochemistry and silver enhancement. An acceptable balance for 28S rRNA ISH detection was obtained using mild cross-linking fixation followed by treatment with a relative high concentration of pepsin for a short time. The ISH method presented in this study was compatible with immunocytochemical detection of protein as demonstrated by double-labeling experiments.     

Ultrastructural localization of mRNA coding for oxytocin by in situ hybridization. Study by high resolution autoradiography using a tritiated oligonucleotide probe

In situ hybridization (ISH) using a 25 mer tritiated oligonucleotide probe has been performed to study at the electron microscopic level the subcellular localization of the oxytocin mRNA in the rat hypothalamic magnocellular neurons. After high resolution radioautography, silver grains appeared to be localized over the cytoplasm of some magnocellular neurons of the supra-optic nucleus and frequently overlapped the ergastoplasmic "cisternae" of the Nissl bodies. These results demonstrate the possible application of ISH at a subcellular level using high resolution radioautography and a tritiated probe.     

Immunocytochemical localization of P0 protein in Golgi complex membranes and myelin of developing rat Schwann cells

P0 protein, the dominant protein in peripheral nervous system myelin, was studied immunocytochemically in both developing and mature Schwann cells. Trigeminal and sciatic nerves from newborn, 7-d, and adult rats were processed for transmission electron microscopy. Alternating 1- micrometer-thick Epon sections were stained with paraphenylenediamine (PD) or with P0 antiserum according to the peroxidase-antiperoxidase method. To localize P0 in Schwann cell cytoplasm and myelin membranes, the distribution of immunostaining observed in 1-micrometer sections was mapped on electron micrographs of identical areas found in adjacent thin sections. The first P0 staining was observed around axons and/or in cytoplasm of Schwann cells that had established a 1:1 relationship with axons. In newborn nerves, staining of newly formed myelin sheaths was detected more readily with P0 antiserum than with PD. Myelin sheaths with as few as three lamellae could be identified with the light microscope. Very thin sheaths often stained less intensely and part of their circumference frequently was unstained. Schmidt-Lanterman clefts found in more mature sheaths also were unstained. As myelination progressed, intensely stained myelin rings became much more numerous and, in adult nerves, all sheaths were intensely and uniformly stained. Particulate P0 staining also was observed in juxtanuclear areas of Schwann cell cytoplasm. It was most prominent during development, then decreased, but still was detected in adult nerves. The cytoplasmic areas stained by P0 antiserum were rich in Golgi complex membranes.     

Cellular localization of a pollen-specific mRNA by in situ hybridization and confocal laser scanning microscopy

Summary The application of confocal laser scanning microscopy together with in situ hybridization experiments in tobacco pollen enabled a detailed localization of a pollen-specific mRNA. The three-dimensional distribution of this specific mRNA over the whole pollen grain was reconstructed by means of optical sections of one specimen.     

Use of a biotinylated DNA probe to detect bacteria transduced by bacteriophage P1 in soil

Presumptive bacteriophage P1 transductants of Escherichia coli, isolated from soil inoculated with lysates of transducing phage P1 and E. coli, were confirmed to be lysogenic for phage P1 by hybridization with a biotinylated DNA probe prepared from the 1.2-kilobase-pair HindIII 3 fragment of bacteriophage P1. No P1 lysogens of indigenous soil bacteria were detected with the DNA probe. The sensitivity and specificity of the DNA probe were assessed with purified and dot blot DNA, respectively. In addition, two techniques for the lysis and deproteinization of bacteria and bacteriophages on nitrocellulose filters were compared. These studies indicated that biotinylated DNA probes may be an effective alternative to conventional radiolabeled DNA probes for detecting specific gene sequences in bacteria indigenous to or introduced into soil.     

Cellular localization of induced human interferon-beta mRNA by non-radioactive in situ hybridization

Induced interferon-beta (IFN-beta) mRNA was localized in human FS-4 fibroblasts by in situ hybridization using biotinylated probes. The hybridization sites were detected by incubation with a nick-translated genomic DNA probe (1.8 kb) via streptavidin-colloidal gold followed by silver contrast enhancement. The positive signals were observed by reflection-contrast light microscopy. IFN-beta mRNA was transiently induced by poly r(I): r(C) in fibroblasts 2-4 h after induction. Induction in the presence of cycloheximide and actinomycin D (superinduction conditions) exhibited an enhanced level of IFN-beta mRNA with a maximum at 4-8 h. The kinetics of the IFN-beta mRNA expression in the cytoplasm as revealed by in situ hybridization proved to be compatible with the results of Northern blotting experiments of total cellular RNA.