Review The Cutting Edge of Histochemical Technique: In Situ PCR
Yoshiki Hiraoka, Motoyuki Ogawa, Yukinao Sakai and Sadakazu Aiso
Department of Anatomy, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582
Received for publication August 8, 1998
The most innovative technique introduced recently into the field of biological study is the polymerase chain reaction (PCR) which can amplify even single-copy DNA sequences to high levels. In situ PCR, which is a method of in situ amplification of DNA of interest in tissue sections, has been subsequently developed and mainly used to investigate tissues infected with viruses. A combination of reverse transcription and PCR has been utilized to detect mRNA of interest in situ. The technique of in situ PCR is promising for a wide variety of applications in basic histology and histopathology.
Key words: DNA amplification, Gene expression, In situ RT/PCR, In situ hybridization
Review Application of Ultrastructural In Situ Hybridization Combined with Immunohistochemistry to Pathophysiological Studies of Pituitary Cell: Technical Review
Akira Matsuno 1, Tadashi Nagashima 1, R. Yoshiyuki Osamura 2 and Keiichi Watanabe 2
1 Department of Neurosurgery, Teikyo University Ichihara Hospital, 3426-3 Anegasaki, Ichihara City, Chiba 299-0111 and 2 Department of Pathology, Tokai University School of Medicine, Boseidai, Isehara City, Kanagawa 259-1100
Received for publication January 17, 1998
In situ hybridization (ISH) at the electron microscopic (EM) level is essential for elucidating the intracellular distribution and role of mRNA in protein synthesis. We describe our EM-ISH method using biotinylated oligonucleotide probes for rat growth hormone (GH) and prolactin (PRL) mRNAs and compare the preembedding method with the postembedding method. The hybridization signal intensity by the postembedding method is lower, and non-specific signals are relatively frequent, in comparison with the preembedding method. The preembedding method thus appears to be easier and better than the postembedding method from the viewpoint of applicability and preservation of mRNA. EM-ISH is considered to be an important tool for clarifying the intracellular localization of mRNA and the exact site of specific hormone synthesis on the rough endoplasmic reticulum. Modulations of ultrastructural mRNA expression induced by stimulatory or inhibitory factors can also be detected with EM-ISH method. The simultaneous visualization of mRNA and encoded protein in the same cells using preembedding EM-ISH and subsequent postembedding immunoreaction with protein A colloidal gold complex is also described. This ultrastructural double staining method for mRNA and encoded protein can be expected to provide an important clue for elucidating the intracellular correlation of mRNA translation and secretion of translated protein.
Key words: In situ hybridization, mRNA, Immunohistochemistry, Electron microscopy, Pituitary cell
Localization of Epidermal Growth Factor Receptor Enhancer Protein in A431 Epidermoid Carcinoma Cells by Southwestern Histochemistry
Ko Komuta 1, Takashi Kanematsu 1, Paul K. Nakane 2 and Takehiko Koji 2
1 Department of Surgery II and 2 Department of Histology and Cell Biology, Nagasaki University School of Medicine, 1-12-4, Sakamoto, Nagasaki 852-8501
Received for publication October 23, 1997
Since abnormal expression of epidermal growth factor receptor (EGFR) is frequently associated with cancer development, the analysis of EGFR gene expression at the transcriptional level as well as the transcript level is helpful to understand the abnormal nature of cancer growth. In this study, we attempted to localize EGFR transcriptional factors, EGFR specific transcription factor (ETF) and GC factor (GCF), in the frozen sections of A431 human epidermoid tumor transplated into nude mice by southwestern histochemistry. As probes for southwestern histochemistry, (+) and (-) sequences of the DNA seqment (91 base pairs (bp)) including ETF and GCF regulatory element were synthesized, allowed to be annealed and then tailed by terminal deoxynucleotidyl transferase with digoxigenin (Dig)-11-dUTP. The sites of Dig were visualized enzymeimmunohistochemically with horseradish peroxidase-labeled anti-Dig. The 91 bp probe detected effectively a single ETF band with a molecular mass of 120 kD on a southwestern blot of the crude nuclear fraction extracted from A431 tumor cells, but not a GCF band. When the frozen sections of A431 tumor were fixed with 4% paraformaldehyde and reacted with the 91 bp probe, the staining of perinuclear area as well as nuclei in a speckled pattern were observed and the staining intensity was increased depending upon the concentrations of the probe and reached a plateu level at 0.5-1 $\mu$g/ml. Moreover, the nuclear staining with the probe was dependent upon a salt concentration and the signal/noise ratio was a maximam at 150 mM NaCl. The staining with the 91 bp probe was abolished by the presence of an excess amount of unlabeled 91 bp DNA or unlabeled ETF responsive element DNA alone, but not by that of unlabeled GCF DNA, indicating that the nuclear and perinuclear staining with the 91 bp probe reflects the localization of ETF. Thus, southwestern histochemistry can be a novel tool to analyze cellular expression of genespecific transcription regulatory factors.
Multiple-Labeling of Oligonucleotide Probes for In Situ Hybridization
Junzo Sasaki 1, Hitoshi Yamamoto 2, Takako Nomura 1, Junko Matsuura 1, Masaharu Seno 3, Eisuke F. Sato 4 and Masayasu Inoue 4
1 Department of Anatomy, Okayama University Medical School, Okayama 700-8558 2 Department of Oral Anatomy, School of Dentistry, Iwate Medical University, Morioka 020-8505, 3 Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Okayama 700-8530 and 4 Department of Biochemistry, Osaka City University Medical School, Osaka 545-8585
Received for publication November 21, 1997
We describe here a method to synthesize probes for in situ hybridization. This method provides more efficient incorporation of the reporter molecules such as 35S-UTP or digoxigenin-UTP into the oligonucleotide probes than other methods.
Two 99-base oligonucleotides complementary to each other were obtained as purified and lyophilized products (>99%). These oligonucleotides were designed as follows. The sequence of 77 bases derived from reported cDNA sequence in the literature was flanked by the restriction sites of EcoR I and Hind III (6 bases for each) with extended random sequences of 5 bases at both ends (total 99 bases). Both oligonucleotides were then annealed and digested with EcoR I and Hind III. The gel-purified EcoR I/ Hind III-cut DNA fragment was cloned into the pGEM4Z vector. The resultant plasmid DNA was linearized with EcoR I or Hind III and used as a template for the synthesis of labeled sense or antisense riboprobes. The amelogenin probes prepared by this method clearly distinguished the localized expression of mRNA when applied to in situ hybridization.
Key words: In situ hybridization, Oligonucleotide probe, Amelogenin
Review Functional Differentiation and its Regulation in Pituitary Cells
R. Yoshiyuki Osamura 1, Naoko Sanno 2, Shigeyuki Tahara 2, Reiko Kurotani 1, Yoshiko Itoh 1 and Akira Teramoto 2
1 Department of Pathology, Tokai University School of Medicine, Isehara-city, Kanagawa 259-1193 and 2 Department of Neurosurgery, Nippon Medical School, Sendagi, Tokyo 113-8602
Received for publication April 14, 1998
It has been an interesting problem to understand why specific hormones are produced from specific cells. It is known that in the human anterior pituitary gland, tumors are classified into those of growth hormone (GH), prolactin (PRL), ACTH, thyroid stimulating hormone (TSH), and gonadotrophin secreting adenomas as well as nonfunctioning adenomas. In human pituitary adenomas, GH secreting adenoma is usually plurihormonal, i.e., the adenoma not only produces GH but also PRL and TSH. TSH secreting adenoma is also plurihormonal, and adenoma produces TSH as well as GH and PRL. Thus, it was supposed that common transcriptional factors participate in the functional differentiation of GH, PRL, and TSH. Pit-1 has been proposed to regulate GH, PRL, and TSH cells and the dwarf mutants in mice were produced by Pit-1 gene mutations. Simmons et al. (1990) observed Pit-1 protein expression in GH, PRL, and TSH cells [10], in the adult rat pituitary gland although Pit-1 mRNA expression was observed in all cell types (Fig. 1). Therefore it was anticipated that, in human pituitary tumors, the functional expression in GH and TSH secreting adenomas may be under the regulation of Pit-1 protein. We describe here the overview of transcriptional factors depicted by immunohistochemistry and in situ hybridization.
Ryohei Katoh, Koichi Suzuki*, Eri Miyagi and Akira Kawaoi
Department of Pathology, Yamanashi Medical University School of Medicine, 1110 Shimokato, Tamaho-cho, Nakakoma-gun, Yamanashi 409-3895 and *Cell Regulation Section, Metabolic Disease Branch, National Institute of Diabetes and Digestion and Kidney Diseases, National Institutes of Health, Bethesda MD 20892, USA
Received for publication May 30, 1998
Thyroid transcription factor 1 (TTF-1), as noted earlier, is a ``thyroid specific'' transcription factor that is a homeodomaincontaining protein responsible for transcriptional activation of thyroid-specific genes, thyroglobulin (Tg), thyroperoxidase (TPO), and thyrotropin receptor (TSHr) genes, in thyroid follicular cells. Little is known about the histological localization or distribution of TTF-1 protein and mRNA, even though many papers on TTF-1 have been published in the past ten years. Therefore, we decided to examine the expression of TTF-1mRNA in normal and neoplastic thyroid tissues using immunohistochemistry and in situ hybridization as well as molecular techniques such as Northern blot and RT-PCR. In addition, extrathyroidal expression of TTF-1 was also investigated in some organs. In this paper, we reviewed literature and discussed the role of TTF-1 in thyroid hormonogenesis, and presented its precise localization or distribution in normal and neoplastic thyroid tissues. In addition, extrathyroidal expression of TTF-1 is briefly discussed.
Received for publication December 17, 1997 and in revised form April 21, 1998
Multi-photon microscopy uses a different principle in exciting fluorochromes from conventional epi-fluorescence microscopy and confocal laser scanning fluorescence microscopy. In multi-photon microscopy a femto second mode locked laser is used as an illumination light source which emits near infra-red light of high peak power (over kilo watt order) and short pulse (about 100 femto seconds at FWHM) at high repetition rate (about 100 MHz). Under illumination of very high intensity by laser scanning fluorescence microscopy, a non linear effect becomes perceivable. Though near infra-red light is not usually absorbed by commonly used fluorochromes in conventional epi-fluorescence microscopy and confocal laser scanning fluorescence microscopy, intense near infra-red light is absorbed by those commonly used fluorochromes in multi-photon microscopy. This non linear interaction of near infra-red light with the fluorochromes under laser scanning microscope introduces unique benefits into the observations of biological specimens.
Key words: Two-photon excitation, Multi-photon excitation, Two-photon microscopy, Multi-photon microscopy, Non linear microscopy, Femto second mode locked laser
Fluorescence Counter-Staining of Cell Nuclear DNA for Multi-Color Laser Confocal Microscopy
Takeshi Suzuki, Toshiyuki Matsuzaki and Kuniaki Takata
Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, Gunma 371-8512
Received for publication December 26, 1997
Twenty-one nucleic acid-specific fluorochromes were examined for use in cell nuclear counter-staining of specimens observed under a laser confocal microscope equipped with a Krypton/argon laser tube. Six green, three red, and two far-red fluorochromes gave sufficient fluorescence. Among them, SYBR Green I, Pico Green, SYTOX Green, YO-PRO-3, and TO-PRO-3 specifically stained the cell nuclear DNA. Other fluorochromes except POPO-3 stained the cell nuclear DNA and co-stained the cyto plasmic RNA. Conversely, POPO-3 specifically stained the cytoplasmic and nucleolar RNA. Anti-bleaching reagents (PPDA and DABCO) effectively prevented the photobleaching of SYBR Green I, Pico Green, YO-PRO-3, and TO-PRO-3, whereas DABCO accelerated the photobleaching of SYTOX Green. Taking into account the characteristics of these fluorochromes, SYBR Green I, YO-PRO-3, and TO-PRO-3 were best suited for cell nuclear counter-staining as green, red, and far-red fluorochromes, respectively. Especially, TO-PRO-3 seems to be the most useful because its far-red fluorescence enables three-color fluorescence staining by combination with immunofluorescence labeling with fluorescein and rhodamine derivatives.
Intracellular Localization and Transcriptional Activation by the Human Glucocorticoid Receptor-Green Fluorescent Protein (GFP) Fusion Proteins
Hidesato Ogawa* and Kazuhiko Umesono
Department of Biochemistry, Institute for Virus Research, Kyoto University, Kyoto 606-8507 and *School of Biological Sciences, Nara Institute for Science and Technology, Ikoma 630-0101
Received for publication May 25, 1998
The initial event during cellular response to glucocorticoid hormone is rapid nuclear transfer of the cytoplasmic glucocorticoid receptor (GR). In order to monitor this process in a single living cell, we employed green fluorescent protein (GFP)-tagged GR to visuallize intracelluar localization and trafficking of the receptor protein in response to ligand. Making a series of deletion GR mutants labeled with the improved GFP mutant, we mapped the intracellular localization signals on the GR protein. In addition, we provide evidence that screening of GR ligands is possible within a single cell.
Key words: Glucocorticoid receptor, Green fluorescent protein, Intracellular trafficking
Immunoreactivity of Vacuolar H+-ATPase in Human Tissues - Using Polyclonal Rabbit Antibodies against V-ATPase Subunits
Shuxin Hu 1, Hisayoshi Inoue 2, Yoshinori Moriyama 3, Kunihiko Goto 1 and Takashi Sawai 4
1 Department of Pathology, Tohoku University Hospital, Sendai 980-77, 2 Department of Orthopaedics, Tohoku Rosai Hospital, Sendai 980, 3 Marine Biological Laboratory, Graduate Department of Gene Science, Faculty of Sciences, Hiroshima University, Mukaishima, Hiroshima 722 and 4 The First Department of Pathology, Iwate Medical University, Morioka 020-8505
Received for publication February 7, 1998 and in revised form April 20, 1998 and re-revised form June 19, 1998
We immunohistochemically examined tissue distribution and cellular localization of vacuolar H+-ATPase (V-ATPase), which is a proton-translocating ATPase known as an acidifier of cytoplasmic vesicles and vacuoles, using two polyclonal rabbit antibodies against A and B subunits of the V-ATPase purified from bovine chromaffin granules. The intense staining was found in epithelial cells of the renal tubules and the salivary ducts, pancreatic $\alpha$ cells, endocrine cells in colon, macrophages, osteoclasts, and chondrocytes in human tissues, and the following three localization patterns of V-ATPase were found in the examined cells: 1) cytoplasmic pattern with granular and diffuse types, 2) polarized pattern, and 3) diffuse apical cytoplasm pattern. The granular type of cytoplasmic pattern was typically observed with high density in macrophages, $\alpha$ cells of the pancreatic islets and endocrine cells in colon. The diffuse type was often seen in most of ductal cells of exocrine glands, osteoclasts, nerve cells with moderate density, and in chondrocytes with high density, and the polarized pattern was represented by intercalated cells of the renal tubules with high density. The diffuse apical cytoplasm pattern was typically found along the brush border of proximal tubules from moderate to high density.
Key words: V-ATPase, Human tissues, Immunohistochemistry
Localization of Gamma-Glutamyltranspeptidase and Alkaline Phosphatase in Primary Cultures of Fetal Rat Hepatocytes
Kohsuke Chida
Department of Anatomy, Kitasato University, School of Allied Health Sciences, Sagamihara, Kanagawa 228-0829
Received for publication February 9, 1998 and in revised form July 14, 1998
The localization of gamma-glutamyltranspeptidase (GGT) and alkaline phosphatase (ALP) in primary cultures of fetal rat hepatocytes was investigated using enzyme cytochemical and immunocytochemical techniques. When hepatocytes were cultured in a medium supplemented with dexamethasone (DEX) or dimethylsulfoxide (DMSO), both enzymes were observed in the plasma membranes along the intercellular spaces that developed between adjacent hepatocytes. On the other hand, in hepatocytes cultured in the basic medium or in medium supplemented with transforming growth factor$\beta$ (TGF-$\beta$), the enzymes were found in the limited area of the cytoplasm surrounding the nuclei. Connexin-32, a subunit of proteins that comprise gap junctions, was detected along the cell borders in hepatocytes cultured in DEX or DMSO-supplemented medium, but was not found in hepatocytes cultured in the basic medium or in medium supplemented with TGF-$\beta$. However, the DEX and DMSO supplements did not cause any changes in the distribution of microtubules in hepatocytes, and numerous microtubular fibers were observed to extend in a radial pattern from the nuclei to the periphery of the cytoplasm in all hepatocytes, irrespective of medium type. These results indicate that assessment of the location of GGT and ALP may help determine the level of hepatocyte differentiation in cultured rat hepatocytes and that intracellular factors other than microtubules are involved in the transport of these enzymes from the cytoplasm to the plasma membrane.
Key words: Gamma-glutamyltranspeptidase, Alkaline phosphatase, Primary culture, Fetal rat hepatocytes
Intestinal Absorption of Lysozyme Molecules and their Destination, an Immunohistochemical Study on Rat
Satimaru Seno 1, Shinichi Inoue 1, Masahiko Akita 2, Kojun Setsu 2, Yuko Tsugaru 2 and Yoshinori Furuhata 3
1 Shigei Medical Research Institute, 2117 Yamada, Okayama 701-0202, 2 Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihama, Yamashiro-cho, Tokushima 771-8055 and 3 Eisai Co., Ltd., Koishikawa 4-6-10, Bunkyo-ku, Tokyo 112-8088
Received for publication February 13, 1998 and in revised form June 3, 1998
Egg white lysozyme is now used clinically for the therapy of inflammatory diseases of respiratory tracts by oral administration. No definite evidence, however, has been presented to show directly the intestinal absorption of lysozyme molecules keeping the enzymatic activity in situ. We therefore tried to immunohistochemically detect egg white lysozyme on the tissues of the intestines and other organs of rats taken out at varied time intervals after the oral administration of the lysozyme. Observations of these rat tissues revealed the egg white lysozyme on the surface of intestinal epithelial cells and in the core tissues of villi, liver parenchymal cells and renal tubular cells showing the specific reaction to the antibody. The findings indicate that egg white lysozyme molecules can be absorbed through the healthy intestinal mucosa of rat keeping the antigenic specificity without being decomposed into amino acids.
Key words: Lysozyme, Intestinal absorption, Immunohistochemical study
1 Developmental Research Laboratories, Shionogi & Co. Ltd., 3-1-1 Futaba-cho, Toyonaka, Osaka 561-0825, 2 Medical Research Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-0034, 3 First Department of Internal Medicine, Miyazaki Medical College, Kihara, Kiyotake-cho, Miyazaki 889-1601 and 4 National Cardiovascular Center Research Institute, Fujishirodai, Suita, Osaka 565-0873
Received for publication February 20, 1998 and in revised form June 8, 1998 and re-revised form July 15, 1998
Adrenomedullin (AM) is a novel hypotensive peptide isolated from the human pheochromocytoma. In the present study, 125I-AM was intravenously administered to rats and its distribution was examined by autoradiographic (ARG) techniques using wholebody, light microscopic (LM) and electron microscopic (EM) methods. Whole-body-ARG after administration of 125I-AM showed the highest radioactivity in the lung (parenchyma), followed by the renal cortex and the liver. When localization of 125I-AM in the lung was examined by LM-ARG, silver grains were localized over the arteries, capillaries and venules. In the arteries, the endothelial cells of the small arteries (lumen diameter 20-50 $\mu$m) were preferentially labelled and there was little labelling on the large arteries.
There were few silver grains on the smooth muscle cells along the entire pulmonary vasculature system. EM-ARG showed many silver grains on the endothelial cells of capillaries and venules. Very few silver grains were observed on the lung when an excess amount of unlabelled AM was administered with 125I-AM.
These results indicated that 125I-AM specific binding sites are most abundant in the lung, and the major binding sites are on the endothelial cells of small arteries, alveolar capillaries and venules.
Department of Pathology, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-0054
Received for publication March 11, 1998 and in revised form July 3, 1998
We investigated the expression of c-jun and c-fos proto-oncogenes and their distribution in the brain of newborn to adult rats to determine the relationship between protooncogene expression and brain development, by the reverse transcription-polymerase chain rection (RT-PCR) method, in situ hybridization and immunohistochemistry. In the RT-PCR analysis, the cDNA fragments of c-jun and c-fos were amplified with expected-sizes, and the expression levels were fixed through all ages examined. The in situ hybridization showed that the expression of c-jun and c-fos mRNA was located at the cytoplasm of most neurons and some glial cells of the entire portions of the cerebrum, brainstem and cerebellum until 300 days after birth. c-Jun protein immunoreactivity was detected in the nucleus of neurons which demonstrated a positive signal by in situ hybridization. Our results suggest that even after neuroblasts differentiate to mature neurons, c-jun and c-fos genes may have a role in the preservation of the neuronal function of the rat central nervous system.
Key words: c-jun, c-fos, Brain development, RT-PCR, In situ hybridization
1 Department of Anatomy and Cell Biology, The University of Tokushima School of Medicine, Tokushima 770-8503 and 2 Department of Biochemistry, Akita University School of Medicine, Akita 010-8543
Received for publication March 20, 1998 and in revised form July 27, 1998
It has been suggested that fetal Leydig cells in rat testes decrease in number just before or soon after birth. However, recent studies have indicated that during the perinatal period, the total number of the Leydig cells does not significantly change. The present study examined apoptotic cell death in the rat testis from embryonic day 18 to postnatal day 49. Apoptotic cells were examined by immunohistochemistry with an antibody against single-stranded DNA (ssDNA), and in situ terminal deoxynucleotidyl transferase staining (in situ TdT). During the perinatal period, ssDNA-immunopositive cells and in situ TdT-positive cells were scattered in the interstitium but were barely present in the seminiferous tubules. After postnatal day 7, the positive cells were abundant in the seminiferous tubules but scarce in the interstitium. Double-immunostaining for ssDNA and a steroidogenic enzyme, P450c17, demonstrated that parts of P450c17positive Leydig cells were ssDNA-positive. Electron microscopic observations revealed that in situ TdT-positive cells had characteristic features of Leydig cells. These results suggest that fetal Leydig cells undergo apoptosis during the perinatal period, and that the number of Leydig cells is regulated by both the rate of cell proliferation and apoptotic cell death.
Key words: Rat testis, Apoptosis, Single-stranded DNA, In situ TdT, Electron microscopy