Four-Hour Double Staining for In Situ Hybridization and Immunohistochemistry

2000 ◽  
Vol 23 (4) ◽  
pp. 321-325 ◽  
Author(s):  
Benio Tsuchiya ◽  
Yuichi Sato ◽  
Kathleen T. Montone ◽  
Tatsuo Nagai ◽  
Toru Kameya
Keyword(s):  
In Situ Hybridization ◽  
Double Staining

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.

A Double Staining Method for Histone H3 mRNA and p53 Protein in Oral Tumors Using In Situ Hybridization and Immunohistochemistry.

1999 ◽  
Vol 32 (4) ◽  
pp. 327-332 ◽  
Author(s):  
Tetsunari Nishikawa ◽  
Shoichi Arai ◽  
Kenichi Uobe ◽  
Masahiro Wato ◽  
Kazuya Tominaga ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Staining Method ◽  
P53 Protein ◽  
Histone H3 ◽  
Double Staining ◽  
Double Staining Method ◽  
Oral Tumors

scholarly journals Sensitive detection of rat gastrin mRNA by in situ hybridization with chemically biotinylated oligodeoxynucleotides: validation, quantitation, and double-staining studies.

1993 ◽  
Vol 41 (2) ◽  
pp. 157-163 ◽  
Author(s):  
L I Larsson ◽  
D M Hougaard
Keyword(s):  
In Situ Hybridization ◽  
Detection System ◽  
Northern Blotting ◽  
Model Systems ◽  
Double Staining ◽  
Adult Rats ◽  
Gastrin Cells ◽  
Human Gastrin ◽  
Almost All

Chemically biotin-labeled oligonucleotides form attractive reagents, as large quantities of stable and well-defined probes can easily be produced. Their usefulness for in situ hybridization was tested using rat gastrin cells as a model. Two probes recognizing two different regions of rat gastrin mRNA were synthesized and produced specific and equally strong hybridization signals. A probe complementary to human gastrin mRNA, but with mismatches to the rat gastrin mRNA sequence, failed to reveal rat gastrin cells under the stringency conditions used. Northern blotting revealed that the rat gastrin mRNA probes reacted exclusively with the appropriately sized (approximately 650 bases) mRNA. Model systems demonstrated that the hybridization signal, as revealed by alkaline phosphatase-based detection, varied linearly with the 10logarithm of target concentration and also showed that a new detection system was much more sensitive than previously used systems. In agreement with previous biochemical data, image analysis showed that starvation of rats led to a progressive decrease in cell staining intensities and cell numbers. Double staining for rat gastrin mRNA and gastrin immunoreactivity showed that in adult rats almost all gastrin cells expressed both mRNA and protein. Similar studies on developing rat gastrin cells revealed discrepancies between gastrin mRNA and gastrin-immunoreactive cells during the first week of newborn life. Subsequently, expression of mRNA and protein in the cells became gradually more concordant.

scholarly journals Staining of the Midbody by an Anti-digoxin-specific Antibody

1998 ◽  
Vol 46 (6) ◽  
pp. 779-782
Author(s):  
Roeland W. Dirks ◽  
Anton K. Raap
Keyword(s):  
In Situ Hybridization ◽  
Specific Antibody ◽  
Cultured Cells ◽  
Cytoplasmic Localization ◽  
Double Staining ◽  
Cytoplasmic Component ◽  
Daughter Cells ◽  
Tubulin Antibodies ◽  
Rna In Situ Hybridization

Using RNA in situ hybridization to reveal cytoplasmic localization patterns of mRNAs in cultured cells, we noted unexpected staining of a cytoplasmic component in telophase cells. Control experiments revealed that the anti-digoxin-specific antibody was responsible for this staining. Because the staining was observed only at a position where both daughter cells are still connected, we identified the stained component as the midbody. This was confirmed by double staining of cells with anti-digoxin and antia -tubulin antibodies. We concluded that anti-digoxin-specific antibody shows crossreactivity with a component present in the midbody.

scholarly journals Simultaneous Detection of RNA and Protein by In Situ Hybridization and Immunological Staining

2001 ◽  
Vol 49 (9) ◽  
pp. 1177-1182 ◽  
Author(s):  
Hideyuki Nagaso ◽  
Takehide Murata ◽  
Noel Day ◽  
Kazunari K. Yokoyama
Keyword(s):  
In Situ Hybridization ◽  
Cell Membranes ◽  
Simultaneous Detection ◽  
Imaginal Discs ◽  
Proteinase K ◽  
Double Staining ◽  
Tissue Integrity ◽  
Specific Proteins

Proteinase K is widely used in methods for detection of transcripts in biological specimens by in situ hybridization (ISH). However, treatment with proteinase K hampers detection of RNA and protein simultaneously. We have developed a method for double staining of transcripts and proteins by ISH and IHC staining in imaginal discs and embryos of Drosophila. Instead of treatment with proteinase K, samples are treated with ethanol plus xylene and with acetone. Acetone renders cell membranes permeable to probes and antibodies without damaging tissue integrity, whereas treatment with proteinase K sometimes damages tissues. Treatment of samples with acetone allows hybridization of probe with transcripts in tissue. It is also effective for immunological staining of samples after ISH with a riboprobe. Thus, our method allows detection not only of transcripts but also of specific proteins in relatively intact single samples. (J Histochem Cytochem 49:1177–1182, 2001)

DNA sequence mapping in interphase and metaphase chromosomes by fluorescence in situ hybridization

1992 ◽  
Vol 50 (1) ◽  
pp. 496-497
Author(s):  
Barbara Trask ◽  
Susan Allen ◽  
Anne Bergmann ◽  
Mari Christensen ◽  
Anne Fertitta ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Dual Band ◽  
Nick Translation ◽  
Metaphase Chromosomes ◽  
Band Pass ◽  
Texas Red ◽  
Fluorescent Spot

Using fluorescence in situ hybridization (FISH), the positions of DNA sequences can be discretely marked with a fluorescent spot. The efficiency of marking DNA sequences of the size cloned in cosmids is 90-95%, and the fluorescent spots produced after FISH are ≈0.3 μm in diameter. Sites of two sequences can be distinguished using two-color FISH. Different reporter molecules, such as biotin or digoxigenin, are incorporated into DNA sequence probes by nick translation. These reporter molecules are labeled after hybridization with different fluorochromes, e.g., FITC and Texas Red. The development of dual band pass filters (Chromatechnology) allows these fluorochromes to be photographed simultaneously without registration shift.

Ultrastructural analysis of the spatial distribution of MRNA

1992 ◽  
Vol 50 (1) ◽  
pp. 552-553
Author(s):  
Gary Bassell ◽  
Robert H. Singer
Keyword(s):  
Electron Microscopy ◽  
Spatial Distribution ◽  
In Situ Hybridization ◽  
High Resolution ◽  
Nucleic Acids ◽  
Colloidal Gold ◽  
Dna Probe ◽  
Nucleotide Analogs ◽  
Gold Particles

We have been investigating the spatial distribution of nucleic acids intracellularly using in situ hybridization. The use of non-isotopic nucleotide analogs incorporated into the DNA probe allows the detection of the probe at its site of hybridization within the cell. This approach therefore is compatible with the high resolution available by electron microscopy. Biotinated or digoxigenated probe can be detected by antibodies conjugated to colloidal gold. Because mRNA serves as a template for the probe fragments, the colloidal gold particles are detected as arrays which allow it to be unequivocally distinguished from background.

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