Multiple-Labeling of Oligonucleotide Probes for In Situ Hybridization.

Sasaki, Junzo; Yamamoto, Hitoshi; Nomura, Takako; Matsuura, Junko; Seno, Masaharu; Sato, Eisuke F.; Inoue, Masayasu

doi:10.1267/ahc.31.275

Cited by 5 publications

(4 citation statements)

References 10 publications

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“…To prepare the antigen, we used synthetic complementary oligodeoxyribonucleotides designed to produce a double-stranded DNA flanked by the restriction enzyme sites, which was then ligated with the bacterial expression vector to produce recombinant oligopeptides. Similar use of oligodeoxyribonucleotides was introduced by Sasaki et al [12] to produce labeled ribonucleotide probes for in situ hybridization. The recombinant oligopeptide adopted in the present method possesses advantages of both the recombinant full-length protein and the synthetic oligopeptide.…”

Section: Discussionmentioning

confidence: 99%

A Time- and Cost-Saving Method of Producing Rat Polyclonal Antibodies

Wakayama

Kato

Utsumi

et al. 2006

Acta Histochem. Cytochem

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Producing antibodies usually takes more than three months. In the present study, we introduce a faster way of producing polyclonal antibodies based on preparation of the recombinant oligopeptide as antigen followed by immunization of rats. Using this method, we produced antisera against two mouse proteins: ERGIC-53 and c-Kit. An expression vector ligated with a pair of complementary synthetic oligodeoxyribonucleotides encoding the protein was introduced into bacteria, and the recombinant oligopeptide fused with the carrier protein glutathione-S-transferase was purified. Wistar rats were immunized by injecting the emulsified antigen subcutaneously into the hind footpads, followed by a booster injection after 2 weeks. One week after the booster, the sera were collected and examined for the antibody titer by immunohistochemistry. Antisera with 1600-fold titer at the maximum were obtained for both antigens and confirmed for their specificity by Western blotting. Anti-ERGIC-53 antisera recognized acinar cells in the sublingual gland, and anti-c-Kit antisera recognized spermatogenic and Leydig cells in the testis. These antisera were applicable to fluorescent double immunostaining with mouse monoclonal or rabbit polyclonal antibodies. Consequently, this method enabled us to produce specific rat polyclonal antisera available for immunohistochemistry in less than one month at a relatively low cost.

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Section: Discussionmentioning

confidence: 99%

A Time- and Cost-Saving Method of Producing Rat Polyclonal Antibodies

Wakayama

Kato

Utsumi

et al. 2006

Acta Histochem. Cytochem

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“…Because they could be readily obtained from a com- mercial source, we chose to conduct this study using oligonucleotide probes: earlier studies had shown that ISH could be successfully carried out using such oligonucleotide probes [6,11,14]. The sequence data of selected genes were obtained from the GenBank internet site, and the sequences of the probes are designed using commercially available computer software.…”

Section: Discussionmentioning

confidence: 99%

Detection of mRNA of Kinesin Superfamily 3A in the Cerebrum and Cerebellum. Biotin-Tyramine-Catalyzed Signal Amplification for In Situ Hybridization.

Takahashi

Kitamura

Tajima

1999

Acta Histochem. Cytochem

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“…The testes were then perfused with Bouin’s fixative and immersed in the same fixative for more than 6 hr, dehydrated in ethanol, substituted with xylene, and embedded in paraffin. Paraffin sections (5 µm thick) were mounted on silane-coated slides [31]. Serial sections were mounted on several sets of three slides.…”

Section: Methodsmentioning

confidence: 99%

Quantification of PERF 15 mRNA in Tissue Sections from Rat Testes

Kogami

Miki

Yamada

et al. 2006

Acta Histochem. Cytochem

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We previously conducted basic research to quantify in situ hybridization (ISH) signals in rat testes. In this experimental model, we selected ribosomal RNA (rRNA) as the hybridizable RNA in paraffin sections, since it allowed us to easily analyze ISH signals expressed with digoxygenin (DIG)-labeled probes quantitatively through “posterization” of the images. We applied this method to analyze the quantification of transcript, PERF 15 mRNA. PERF 15 is expressed specifically in the testes and localized in the rigid cytoskeletal structure of the sperm head, and has been considered to be involved in the apoptotic process of spermatogenic cells. Quantification of the signals may help to clarify the detailed function of PERF 15. We further analyzed the signals concomitant with a confocal laser scanning microscope. The peak of PERF 15 mRNA expression was found in diplotene spermatocytes, and the amount of PERF 15 mRNA was greatest in late pachytene and diplotene spermatocytes and early spermatids, followed by early pachytene spermatocytes, and then late spermatids. PERF 15 may be involved in the events leading to meiotic division, in which apoptosis is also involved. The present study may help to determine the concentration of mRNA in tissue sections.

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Multiple-Labeling of Oligonucleotide Probes for In Situ Hybridization.

Cited by 5 publications

References 10 publications

A Time- and Cost-Saving Method of Producing Rat Polyclonal Antibodies

A Time- and Cost-Saving Method of Producing Rat Polyclonal Antibodies

Detection of mRNA of Kinesin Superfamily 3A in the Cerebrum and Cerebellum. Biotin-Tyramine-Catalyzed Signal Amplification for In Situ Hybridization.

Quantification of PERF 15 mRNA in Tissue Sections from Rat Testes

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