Nonradioactive in situ Hybridization Techniques for Routinely Prepared Pathology Specimens and Cultured Cells

1994 ◽  
Vol 17 (4) ◽  
Author(s):  
Gan
Keyword(s):  
In Situ Hybridization ◽  
Cultured Cells ◽  
Nonradioactive In Situ Hybridization

scholarly journals Detection of v- and c-erbB oncogene mRNA in cultured cells by in situ hybridization.

1990 ◽  
Vol 52 (1) ◽  
pp. 175-178
Author(s):  
Toshio IKEDA ◽  
Yasuhiro YOSHIKAWA ◽  
Kazuya YAMANOUCHI
Keyword(s):  
In Situ Hybridization ◽  
Cultured Cells

Methodologies for specific intron and exon RNA localization in cultured cells by haptenized and fluorochromized probes

1993 ◽  
Vol 104 (4) ◽  
pp. 1187-1197 ◽  
Author(s):  
R.W. Dirks ◽  
F.M. van de Rijke ◽  
S. Fujishita ◽  
M. van der Ploeg ◽  
A.K. Raap
Keyword(s):  
In Situ Hybridization ◽  
Cell Lines ◽  
Cultured Cells ◽  
Direct Detection ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Immediate Early ◽  
High Signal ◽  
Pretreatment Conditions

We have determined optimal conditions for the detection of mRNA sequences in cultured cells by nonradioactive in situ hybridization. For this purpose a number of different cell lines have been used: rat 9G cells for the detection of human cytomegalovirus immediate early mRNA, and HeLa as well as 5637 carcinoma cells for the detection of housekeeping gene mRNAs. Extensive optimization of fixation and pretreatment conditions revealed that most intense hybridization signals are obtained when cells are grown on glass microscope slides, fixed with a mixture of formaldehyde and acetic acid, pretreated with pepsin and denatured prior to hybridization. In addition, we also studied the potential of fluorochromized probes for the direct detection of multiple RNA sequences. The optimized in situ hybridization procedure revealed that immediate early mRNA transcripts are, in addition to a cytoplasmic localization, localized within nuclei of rat 9G cells. Double hybridization experiments showed that intron and exon sequences colocalize within the main nuclear signal. In addition, the presence of small, intron-specific, fluorescent spots scattered around the main nuclear signals indicates that intron sequences which are spliced out can be visualized. Additional information about the functioning of cells could be obtained by the detection of mRNA simultaneously with bromodeoxyuridine, incorporated during S-phase, or its cognate protein. The sensitivity of these methods is such that mRNAs of abundantly expressed housekeeping genes can be detected in a variety of cell lines with high signal to noise ratios.

Nonradioactive in Situ Hybridization with Digoxigenin Labeled DNA Probes

1992 ◽  
Vol 67 (2) ◽  
pp. 59-67 ◽  
Author(s):  
N. Arnold ◽  
R. Seibl ◽  
C. Kessler ◽  
J. Wienberg
Keyword(s):  
In Situ Hybridization ◽  
Dna Probes ◽  
Nonradioactive In Situ Hybridization

scholarly journals Freeze-drying Allows Double Nonradioactive ISH and Antigenic Labeling

2000 ◽  
Vol 48 (4) ◽  
pp. 499-507 ◽  
Author(s):  
Huguette Louis ◽  
Julie Lavie ◽  
Patrick Lacolley ◽  
Danièle Daret ◽  
Jacques Bonnet ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Freeze Drying ◽  
Simultaneous Detection ◽  
Tissue Preservation ◽  
Reliable Technique ◽  
Mrna Detection ◽  
Nonradioactive In Situ Hybridization

Because tissue freeze-drying is an excellent way to preserve antigenic conformation, we have tested the feasibility of this technique to reveal nonradioactive in situ hybridization (ISH) of tissue mRNA. We have compared mRNA detection after different methods of tissue preservation, freeze-drying, cryosectioning, and formaldehyde or methanol fixation. Our results show that nonradioactive ISH is more sensitive for tissues preserved by freeze-drying than for other tissue preparations. We have demonstrated that freeze-drying allows combination of ISH and immunohistochemistry for simultaneous detection of mRNA and antigen because with this technique of tissue preservation ISH does not affect the sensitivity or the amount of the detected antigens. This work underscores the fact that tissue freeze-drying is an easy, convenient, and reliable technique for both ISH and immunohistochemistry and achieves excellent structural conditions for nonradioactive detection.

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