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Volume 26 (1993)
- Issue 6 Pages 507-
- Issue 5 Pages 337-
- Issue 4 Pages 251-
- Issue 3 Pages 165-
- Issue 2 Pages 75-
- Issue 1 Pages 1-
Volume 26, Issue 4
Displaying 1-10 of 10 articles from this issue
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SOTOKICHI MORII, NOBUAKI SHIKATA, IEZO NAKAO1993 Volume 26 Issue 4 Pages 251-259
Published: 1993
Released on J-STAGE: October 28, 2009
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SADAKI YOKOTA1993 Volume 26 Issue 4 Pages 263-273
Published: 1993
Released on J-STAGE: October 28, 2009
JOURNAL FREE ACCESSSeveral basic problems in quantitative immunoelectron microscopy were investigated. The catalase activities in the liver homogenate of guinea pig, rat, Chinese hamster and mouse were assayed biochemically, while the peroxisomal labeling density (gold particles/μm2) for this enzyme of the same animals was determined by protein A-gold technique. The species difference of the enzyme activity did not correspond to the difference of the labeling density. Furthermore, the difference of the enzyme activity did not correspond to the staining intensity of the signal developed by immunoblot for catalase. However, the labeling density largely corresponded to the immunoblot data. Peroxisomal labeling densities of the above animals were determined by protein A-gold technique using rabbit anti-rat liver catalase (RLC) or guinea pig anti-RLC. The data obtained by the rabbit antibody was not consistent with those obtained by the guinea pig antibody. Moreover, we investigated the relationship between antigen concentration and labeling density using a model system in which bovine serum albumin (BSA) or purified RLC is directly mixed with LR White resin at various concentrations. The labeling density was directly proportional to the antigen concentration within the examined range. The results clearly demonstrate that the antigen concentration is expressed by the labeling density. Such proportionality is realized only between the same species. Taken altogether, we could compare data obtained quantitative immunoelectron microscopy between the same species even if using the same lot of antibody.View full abstractDownload PDF (12888K) -
YUTAKA TASHIRO, AKITSUGU YAMAMOTO1993 Volume 26 Issue 4 Pages 275
Published: 1993
Released on J-STAGE: October 28, 2009
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JUN WATANABE1993 Volume 26 Issue 4 Pages 277-279
Published: 1993
Released on J-STAGE: October 28, 2009
JOURNAL FREE ACCESSIn this paper, two quantitative light microscopic immunohistochemical methods are reported. One is the method for measuring an antigen content in tissue sections by a combination of enzyme immunohistochemistry with microphotometry and nitrocellulose (NC) binding assay. The NC model systems used in this method gave quantitative information on the relation between the amount of antigen and the intensity of immunostaining, and provided the apparent extinction coefficient. The other is the method using video-image processing. This method can measure an antigen content in sections rapidly and easily, even if the antigen could not be immobilized on NC membrane. These two methods are unable to measure all antigen contents in tissue sections.View full abstractDownload PDF (502K) -
TETSUJI NAGATA1993 Volume 26 Issue 4 Pages 281-291
Published: 1993
Released on J-STAGE: October 28, 2009
JOURNAL FREE ACCESSIn order to quantify histochemical reactions, light and electron microscopic radioautograms obtained from mouse organs were analyzed by means of various kinds of available image analyzers. Cultured cells in vitro or epithelial cells of the stomachs, small and large intestines, the livers and the pancreases of mice aged from fetal day 19 to postnatal 2 years were labeled with RI-labeled precursors for macromolecules, e. g. 3H-thymidine for DNA, 3H-uridine for RNA, 3H-amino acid for proteins, 3H-glucosamines and 35SO4 for mucosubstances, 3H-glycerol for lipids and all the cells and tissues were fixed in glutaraldehyde and osmium tetroxide, embedded in Epon, sectioned either thick (2μm) or thin (0.2μm), coated with either Konica NR-M2 or-H2 radioautographic emulsions, exposed for several weeks or months, developed in either SDX-1 or GL-phenidon developers and stained with either toluidine blue or lead citrate for either light or electron microscopy.
Light microscopic radioautograms (LMRAG) were observed with Olympus Vanox AHB-LB or Nikon Optiphot-2 and analyzed quantitatively with IBAS-II (Carl-Zeiss) or Quadra 900 (Macintosh) for labeling indices and grain counts. Electron microscopic radioautograms (EMRAG) were observed in Hitachi H-700 or JEOL JEM-4000EX electron microscopes at accelerating voltages of 200 or 400kV, and enlarged photographs were analyzed with several kinds of image analyzers, PPA-250 (Rhesca), MOP (Kontron), Digigramer-G (Mutoh Kogyo) and Quadra 900 (Macintosh) for grain counting or measuring the sizes and numbers of cell organelles. Some of EMRAG were analyzed in analytical electron microscopes, combined with Hitachi H-700 Horiba EMAX-1800E, JEOL JEM-200CX Kevex 7000-77, and JEM-4000EX Tracor Northern TN-5400.
From the results, it was demonstrated that correct data can, to some extent, be obtained by controlling both full automatic and semi-automatic analyzers by operating them manually.View full abstractDownload PDF (17968K) -
SOHEI KITAZAWA, RIKO KITAZAWA, CHONG-HUA YAO, SAKAN MAEDA1993 Volume 26 Issue 4 Pages 295-301
Published: 1993
Released on J-STAGE: October 28, 2009
JOURNAL FREE ACCESSIn the current study, we developed an improved method for ISH at the electron microscopic level, in which we employed pre-embedding hybridization using a BrdU labeled probe followed by post-embedding immunoglobuline gold colloid staining (IGS). Because metabolic labeling of BrdU provides an almost evenly labeled DNA probe with high labeling index, pre-embeddingly hybridized BrdU-labeled probe preserves its detectable antigenicity even after the preparation of ultrathin sections. We demonstrated precise cell identification, early stage of gene expression and alternative splicing patterns using this technique.View full abstractDownload PDF (10433K) -
SHINTARO NOMURA, KIMIAKI HIRAKAWA, JUNSUKE NAGOSHI, SEIICHI HIROTA, HY ...1993 Volume 26 Issue 4 Pages 303-309
Published: 1993
Released on J-STAGE: October 28, 2009
JOURNAL FREE ACCESSThe sensitive and high resolution method of in situ hybridization technique has been developed using digoxigenin-11 UTP labeled cRNA as a probe. The system was applied to the decalcified mineralized tissues such as bone and dentin. Using the system, the localization of the mRNA of bone extracellular matrix proteins, osteopontin (Osp), osteonectin (Osn), Osteocalcin (Osc) and matrix Gla protein (MGP) was examined in decalcified bone. Decalcified femurs and mandibulae of embryo, neonatal, 2 to 40-week old mice were used for examination. Osn and Osc mRNAs were localized at osteoblast in bone and at odontoblast in dentin. Although the distribution pattern of Osn positive cells and Osc positive cells was partially overlapped, Osc mRNA was detected at matured osteoblast and odontoblast but Osn mRNA was detected not only at matured but differentiating osteoblast such as flat osteoblast in the periosteal layer. Osp mRNA was detected at osteoblast in bone, but no apparent expression of Osp mRNA was found at odontoblast in dentin. MGP mRNA was detected in hypertrophic chondrocytes. These results indicated the usefulness of this system for identifying the cell types in bone and dentin.View full abstractDownload PDF (4381K) -
TSUYOSHI TAKASU, YASUSHI FURUTA, SEIGO SUZUKI, YOSHIHIKO OBARA, KAZUO ...1993 Volume 26 Issue 4 Pages 311-317
Published: 1993
Released on J-STAGE: October 28, 2009
JOURNAL FREE ACCESSWe applied in situ hybridization (ISH) using RNA probes which hybridize with infected-cell protein number zero (ICP0) mRNA or latency-associated transcripts (LAT) to detect herpes simplex virus type 1 (HSV-1) in both latent and productive infections. In the productive stage, in which the production of viral particles can be shown by immunohistochemistry and electron microscopy, we demonstrated the presence of ICP0 mRNA and LAT, which indicates that LAT may also be transcribed in productive infection. In latent infection of the trigeminal and geniculate ganglia, where HSV antigen cannot be detected by immunohistochemistry, we detected positive hybridization signals using ISH with ICP0 sense RNA probe, which is complementary to LAT. Positive signals were not detected with ICP0 antisense probe. We could not demonstrate HSV latency in the spiral ganglia using ISH, although the HSV genomes were amplified by using the polymerise chain reaction method. These results demonstrate that ISH is a convenient, but limited, technique for the detection of latent HSV.View full abstractDownload PDF (6583K) -
JOHJI INAZAWA, TAKESHI AZUMA, TAKESHI ARIYAMA, TATSUO ABE1993 Volume 26 Issue 4 Pages 319-324
Published: 1993
Released on J-STAGE: October 28, 2009
JOURNAL FREE ACCESSWe developed a simple technique for delineation of refined replication G-band in combination with fluorescent in situ hybridization (FISH). By using this system we localized three genes of pepsinogen C (PGC), renin (REN), and cathepsin E (CTSE) on 6p21.1, 1q32, and 1q32, respectively. Further, we determined the physical order of REN and CTSE on chromosome band 1q32 as cen-(1q32)-REN-CTSE-tel by multi-color FISH.View full abstractDownload PDF (6147K) -
TAKASHI KURAMOTO, MASAYUKI MORI, NOBUYUKI HIRAYAMA, SAKURA SABURI, JUN ...1993 Volume 26 Issue 4 Pages 325-332
Published: 1993
Released on J-STAGE: October 28, 2009
JOURNAL FREE ACCESSTo construct rapidly both genetic and physical maps in the laboratory rat (Rattus norvegicus), we examined the availability of cosmid clones containing rat genomic large DNA inserts as resources of informative markers. Ninety of the 95 cosmid clones examined were specifically hybridized with an (AC)15 oligonucleotide probe, which suggested that they contain (AC/TG)n repeats which are highly polymorphic. Primer sets were prepared to amplify the microsatellite regions by polymerase chain reaction (PCR), and linkage analysis based on the size polymorphisms was successfully carried out. Each DNA insert could be assigned to a particular chromosome by rat×mouse somatic cell hybrid analysis, and localized within a discrete chromosomal subregion by fluorescent in situ hybridization (FISH) using the cosmid probes. This approach should be an excellent strategy for rapid construction of an ideal genetic marker map. In addition, a genomic library consisting of the mapped clones should become a useful tool for positional cloning of causative genes in rat models of human genetic disease.View full abstractDownload PDF (6352K)
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