Oligonucleotide-Primed In Situ Transcription and Immunogold-Silver Staining Systems: Localization of mRNA in Tissues and Cells

1995 ◽  
pp. 99-112
Author(s):  
Lawrence E. De Bault ◽  
Bao-Le Wang
Keyword(s):  
Silver Staining

Detection of atrial natriuretic peptides (ANP) in rat atria by immunogold-silver staining (IGSS)

1994 ◽  
Vol 52 ◽  
pp. 304-305
Author(s):  
Robyn Rufner ◽  
Gerhard W. Hacker ◽  
Michele Forte ◽  
Nancyleigh E. Carson ◽  
Cristina Xenachis ◽  
...  
Keyword(s):  
Natriuretic Peptides ◽  
Silver Staining ◽  
Sodium Thiosulfate ◽  
Atrial Natriuretic Peptides ◽  
Paraffin Sections ◽  
Glass Slides ◽  
Metallic Salts ◽  
Wistar Kyoto ◽  
Right Atria

The use of immunogold-silver staining (IGSS) to enhance label penetration and Localization for immunocytochemistry or in situ hybridization utilizing a variety of metallic salts has been documented. In this morphological study, the effects of silver acetate, silver lactate and silver nitrate were evaluated for immunogold-labeling of a trial natriuretic peptides (ANP) in rat right atria.Eight Wistar Kyoto retired breeders were sedated with pentobarbital, perfused with either 4% paraformaldehyde (LM) or Karnovsky's fixative (EM), and right atria were dissected, processed, embedded in paraffin or epon, respectively and sectioned according to conventional methods. For light microscopy, an indirect IGSS method according to Hacker (3) was performed. Paraffin sections on glass slides were washed in ddH2O, immersed in Lugol's iodine, washed in ddH2O and treated with 2.5% aqueous sodium thiosulfate for 20 sec. After additional washes in ddH2O and TBS-0.1% fish gelatin, 10% normal goat serum (PBS with 1% BSA) was applied for 20 min before an overnight incubation at 4°C with a polyclonal α-ANP primary antibody (Peninsula Labs, 1:1000 in TBS/BSA).

Sequential silver staining and in situ hybridization reveal a direct association between rDNA levels and the expression of homologous nucleolar organizing regions: a hypothesis for NOR structure and function

1998 ◽  
Vol 111 (10) ◽  
pp. 1433-1439
Author(s):  
F. Zurita ◽  
R. Jimenez ◽  
M. Burgos ◽  
R.D. de la Guardia
Keyword(s):  
Transcription Factors ◽  
In Situ Hybridization ◽  
Silver Staining ◽  
Organizer Region ◽  
Nucleolar Organizer ◽  
Interindividual Variation ◽  
Nucleolar Organizer Regions ◽  
Single Pair ◽  
Ribosomal Cistrons

We have developed a procedure for sequential silver staining and in situ hybridization to analyze the relationship between the amount of rDNA present in nucleolar organizer regions, as estimated by in situ hybridization, and their level of expression, as estimated by the silver signal. For simplicity we used cells from the insectivorous mole Talpa occidentalis, which have a single pair of nucleolar organizer regions in chromosome pair 3. The relative content of ribosomal cistrons was also related to the hierarchy of activation of the nucleolar organizer regions present in this chromosomal pair. Statistical analyses demonstrated that both the relative level of expression and the activation hierarchy depended mainly on the number of ribosomal cistrons in nucleolar organizer regions. We propose a functional two-step hypothesis, which is consistent with most known data concerning interchromosomal, intercellular and interindividual variation in a number of plant and animal species, including Talpa occidentalis. In step one, the first available transcription factors bind randomly to the ribosomal promoters, such that larger nucleolar organizer regions are more likely to recruit them. In the second step the remaining transcription factors are recruited in a cooperative way, thus completing activation of one nucleolar organizer region, before the next one becomes active.

Physical localization of active and inactive rRNA gene loci in Hordeum marinum spp. gussoneanum (4x) by in situ hybridization

Genome ◽  
1992 ◽  
Vol 35 (6) ◽  
pp. 1032-1036 ◽  
Author(s):  
Ib Linde-Laursen ◽  
Elly Ibsen ◽  
Roland Von Bothmer ◽  
Henriette Giese
Keyword(s):  
In Situ Hybridization ◽  
Silver Staining ◽  
Chromosome Pair ◽  
Extra Chromosome ◽  
Rrna Gene ◽  
Morphological Similarity ◽  
Identical Position ◽  
Additional Support ◽  
Two Populations

Two populations of the diploid and 10 populations of the tetraploid cytotype of Hordeum marinum ssp. gussoneanum were studied for the presence of chromosomal segments harbouring rDNA. Conventional cytological methods established the presence of only one satellited (SAT) chromosome pair in both cytotypes. This was supported by silver staining revealing two NORs and two standard-sized nucleoli. Two additional micronucleoli were observed in a few interphases of two tetraploid populations indicating the presence of an extra chromosome pair with very low nucleolus-forming activity. In situ hybridization with the wheat rDNA probe pTA71 identified intense signals at the nucleolar constrictions of the SAT chromosomes of both cytotypes and weaker signals in a chromosome pair of the tetraploid cytotype, morphologically similar to the SAT chromosomes but without visible nucleolar constrictions. This confirms the presence of rDNA in two chromosome pairs in the tetraploid cytotype. The morphological similarity between these two pairs and the SAT-chromosome pair of the diploid cytotype as well as an identical position of the signals in all three pairs give additional support to an autoploid origin of the tetraploid cytotype. The rDNA at the nucleolar constrictions consisted of two segments of condensed rDNA of different sizes connected by diffuse rDNA. The rDNA of the chromosome pair without nucleolar constrictions was condensed supporting that this conformation is connected with inactivity. The tripartite structure of the rDNA at the nucleolar constrictions corresponds to similar tripartite structures observed after silver staining and Giemsa C-banding.Key words: in situ hybridization, rDNA location, Hordeum marinum, autoploidy, inactive NORs.

scholarly journals Combined beta-galactosidase and immunogold/silver staining for immunohistochemistry and DNA in situ hybridization.

1990 ◽  
Vol 38 (3) ◽  
pp. 325-329 ◽  
Author(s):  
W van den Brink ◽  
C van der Loos ◽  
H Volkers ◽  
R Lauwen ◽  
F van den Berg ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Silver Staining ◽  
Staining Method ◽  
Double Staining ◽  
Reaction Products ◽  
Silver Enhancement ◽  
Silver Staining Method ◽  
Double Staining Method ◽  
Beta Galactosidase

A combination of beta-galactosidase enzyme and the immunogold/silver staining method was studied for evaluation of double-staining experiments. Applications are shown for immunohistochemical double staining using two monoclonal antibodies and for combined immunohistochemistry and DNA in situ hybridization in one tissue section. The following advantages for the present double-staining method were evaluated: superior sensitivity of the immunogold/silver staining method for at least one epitope, which also allows detection of biotinylated DNA probes. The structure of the indolyl precipitate after revelation of beta-galactosidase activity did not show a concealing effect during a sequential double-staining method, as compared with the visualization of peroxidase with diaminobenzidine. These factors, and the sharply contrasting colored reaction products of beta-galactosidase (blue-green) and the immunogold/silver staining method including silver enhancement (brown-black), allow clear distinction of mixed-stained cell constituents.

In situ hybridization technique using an immunogold silver staining system

1989 ◽  
Vol 21 (7) ◽  
pp. 425-428 ◽  
Author(s):  
P. Jackson ◽  
F. A. Lewis ◽  
M. Wells
Keyword(s):  
In Situ Hybridization ◽  
Silver Staining ◽  
Hybridization Technique

scholarly journals Karyotypic variation in the long-whiskered catfish Pimelodus blochii Valenciennes, 1840 (Siluriformes, Pimelodidae) from the lower Tapajós, Amazonas and Trombetas Rivers

2018 ◽  
Vol 12 (3) ◽  
pp. 285-298 ◽  
Author(s):  
Ivanny Coelho da Fonseca ◽  
Luan Aércio Melo Maciel ◽  
Frank Raynner Vasconcelos Ribeiro ◽  
Luís Reginaldo Ribeiro Rodrigues
Keyword(s):  
Silver Staining ◽  
Chromosome Pair ◽  
Fluorescent In Situ Hybridization ◽  
Species Complex ◽  
Diploid Number ◽  
Acrocentric Chromosome ◽  
5S Rdna ◽  
Karyotypic Formula ◽  
Karyotypic Variation

The genus Pimelodus LaCépède, 1803 comprises 35 formally recognized species distributed along the major neotropical river basins. Despite conservatism in diploid number with 2n=56, an intense variation of chromosomal morphology (karyotypic formula) has been documented in Pimelodus species. In the present study, we analyzed karyotypes of 20 specimens, identified as Pimelodusblochii Valenciennes, 1840 and collected from the lower courses of the Tapajós, Amazonas and Trombetas Rivers. The karyotypes were characterized by Giemsa conventional staining, C-banding, silver staining (Ag-NOR) and fluorescent in situ hybridization (FISH) with 5S and 18S rDNA probes. The karyotypes showed 2n=56 chromosomes in fish from the Tapajós River. In contrast, fish from the Amazonas and Trombetas Rivers had 2n=58. The nucleolus organizing regions were labeled on the short arm of an acrocentric chromosome as demonstrated by silver staining and FISH. Signals for 18S and 5S rDNA were co-localized on one chromosome pair. Our results demonstrate karyotypic divergence between Tapajós and Amazonas-Trombetas populations of P.blochii, interpreted as supporting the existence of a species complex in this taxon.

Sensitive Detection of DNA and mRNA Sequences by In Situ Hybridization and Immunogold-Silver Staining

1995 ◽  
pp. 113-130
Author(s):  
Gerhard W. Hacker ◽  
Ingeborg Zehbe ◽  
Cornelia Hauser-Kronberger ◽  
Jiang Gu ◽  
Angelika Graf ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Silver Staining ◽  
Sensitive Detection
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