[24] In Situ hybridization and immunodetection techniques for simultaneous localization of messenger RNAs and protein epitopes in tissue sections and cultured cells

We present here a method enabling the simultaneous detection of two messenger RNAs in tissue sections by use of a two-step in situ hybridization procedure. Tissue sections were hybridized with a radioactive probe and coated with emulsion. The emulsion was processed for development, fixed, and a second hybridization was performed through the emulsion with a biotinylated probe subsequently revealed with streptavidin-alkaline phosphatase. This procedure allows the detection of two mRNAs without loss of signal, removal of the emulsion, or spurious reaction. The simultaneous detection of oxytocin and vasopressin mRNAs in the hypothalamus, and of dopamine receptor and neuropeptide mRNAs in the striatum, demonstrated the efficiency of the procedure. Such a two-step procedure provides a simple and flexible way to make possible comparative analysis of the localization of two mRNAs within the same tissue section.

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Co-localization of EGF transcripts and peptides by combined immunohistochemistry and in situ hybridization.

Journal of Histochemistry & Cytochemistry ◽

10.1177/38.12.2254649 ◽

1990 ◽

Vol 38 (12) ◽

pp. 1853-1857 ◽

Cited By ~ 14

Author(s):

R I Couwenhoven ◽

W Luo ◽

M L Snead

Keyword(s):

In Situ Hybridization ◽

Growth Factor ◽

Simultaneous Detection ◽

Interstitial Cells ◽

Glandular Epithelium ◽

Messenger Rnas ◽

Tissue Sections ◽

Representative Model ◽

Epidermal Growth

There is increasing evidence that autocrine- and paracrine-acting growth factors participate in cell and tissue development, maintenance, and renewal. Recent advances in histochemical techniques have facilitated the localization of growth factor messenger RNAs or polypeptides in tissue sections. However, the spatial relationships between the sites of growth factor transcription, translation, and post-translational processing to functional bioactive peptides have been difficult to correlate because each method of detection requires separate tissue sections. We undertook the simultaneous detection of epidermal growth factor (EGF) transcripts and EGF epitopes by combining immunohistochemistry methods with in situ hybridization. Adult mouse submandibular gland was chosen as a representative model because it contains sites of EGF biosynthesis which may participate in mediating the development, maintenance, and renewal of the organ through autocrine or paracrine mechanism(s). Granular duct (GD) cells demonstrated the presence of both EGF transcripts and EGF peptides. In contrast, the interstitial cells lying adjacent to glandular epithelium also contained relatively high levels of EGF transcripts, although no mature EGF peptides were detected. The experimental approach of co-localization and the resulting data indicate previously unreported sites of EGF transcription in glandular interstitial cells, which may provide molecular information required for the morphogenesis and differentiation of adjacent glandular epithelium.

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

The Japanese Journal of Veterinary Science ◽

10.1292/jvms1939.52.175 ◽

1990 ◽

Vol 52 (1) ◽

pp. 175-178

Author(s):

Toshio IKEDA ◽

Yasuhiro YOSHIKAWA ◽

Kazuya YAMANOUCHI

Keyword(s):

In Situ Hybridization ◽

Cultured Cells

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

Journal of Cell Science ◽

10.1242/jcs.104.4.1187 ◽

1993 ◽

Vol 104 (4) ◽

pp. 1187-1197 ◽

Cited By ~ 5

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.

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