Postnatal ontogeny of POMC gene expression in the rat pituitary: an analysis by in situ hybridization histochemistry

We have developed a method of non-radioactive in situ hybridization histochemistry using alkaline phosphatase-labeled oligonucleotide probes to detect gene expression in the intestine. Because the intestine contains a large amount of endogenous alkaline phosphatase activity, mild acid pretreatment of the tissue sections was required to inactivate the alkaline phosphatase. Acid pre-treatment dramatically reduced the endogenous activity without affecting the efficiency of hybridization or the probe's ability to reveal a positive mRNA signal. Furthermore, the addition of polyvinyl alcohol to the substrate solution helped to keep the background staining low without adversely affecting the intensity of the signal. The current protocol allows rapid and sensitive detection of sites of gene expression in intestinal tissue.

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Quantitative in-situ hybridization histochemistry studies on growth hormone (GH) gene expression in acromegalic somatotrophs: effects of somatostatin, GH-releasing factor and cortisol

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0010019 ◽

1988 ◽

Vol 1 (1) ◽

pp. 19-26 ◽

Cited By ~ 17

Author(s):

A. Levy ◽

S. L. Lightman

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

In Situ Hybridization ◽

Α Subunit ◽

Hybridization Histochemistry ◽

Subunit Mrna ◽

In Situ Hybridization Histochemistry ◽

Gh Gene ◽

Dispersed Cells

ABSTRACT We have examined the effects of human GH-releasing factor (1–44) (GRF), cortisol and somatostatin-(1–14) on GH gene expression in solid tissue and dispersed cells from human pituitary adenomas using quantitative in-situ hybridization histochemistry. Sections cut from tissue obtained at hypophysectomy from three acromegalic patients were hybridized to probes directed against mature α-subunit, GH, prolactin, pro-opiomelanocortin, TSHβ-subunit and LHβ-subunit mRNA. Only one biopsy contained GH mRNA in isolation. A second was found to co-exhibit GH, prolactin and α-subunit mRNA, and a third was found to contain prolactin, TSHβ-subunit, α-subunit and LHβ-subunit mRNA, with GH mRNA below the limit of specific detection, indicating that the sample was composed of normal rather than adenomatous pituitary tissue. GH mRNA in individual dispersed cells derived from the latter declined to barely detectable levels over 287 h, both in cultures containing GRF (10 ng/ml) or GRF (10 ng/ml) plus somatostatin (10 ng/ml) and in controls, but increased fourfold in cultures containing GRF (10 ng/ml) plus cortisol (0·5 μmol/l). GH mRNA remained unchanged in both adenoma samples over 138 and 450 h, irrespective of the addition of GRF or GRF plus hydrocortisone. In these samples, somatostatin plus GRF had no consistent effect. These studies confirm that quantitative in-situ hybridization histochemistry can be used to investigate hormone gene regulation in small samples of human tissue and should enable us to define more clearly the level at which abnormal gene regulation occurs.

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