scholarly journals An in situ hybridization histochemistry method for the use of alkaline phosphatase-labeled oligonucleotide probes in small intestine.

1991 ◽  
Vol 39 (10) ◽  
pp. 1377-1384 ◽  
Author(s):  
H Kiyama ◽  
P C Emson
Keyword(s):  
Gene Expression ◽  
Alkaline Phosphatase ◽  
In Situ Hybridization ◽  
Oligonucleotide Probes ◽  
Acid Pretreatment ◽  
Hybridization Histochemistry ◽  
In Situ Hybridization Histochemistry ◽  
Pre Treatment ◽  
Endogenous Activity

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.

In situ hybridization histochemistry of mRNAs for hormones and chromogranins in normal pituitary tissue and pituitary adenoma

1991 ◽  
Vol 125 (6) ◽  
pp. 628-636 ◽  
Author(s):  
Ferenc Kilár ◽  
Carin Muhr ◽  
Keiko Funa
Keyword(s):  
In Situ Hybridization ◽  
Pituitary Gland ◽  
Chromogranin A ◽  
Oligonucleotide Probes ◽  
Chromogranin B ◽  
Hybridization Histochemistry ◽  
In Situ Hybridization Histochemistry ◽  
Normal Pituitary Gland ◽  
Positive Hybridization

Abstract. In situ hybridization histochemistry was employed to detect mRNAs of pituitary hormones and chromogranins in normal pituitary gland and pituitary adenomas. Oligonucleotide probes specific to the mRNAs for prolactin, growth hormone, proopiomelanocortin, the α- and •-subunits of the glycoprotein hormones and chromogranins A and B were used in the hybridization experiments. The oligonucleotides of 27 to 51 bases were labelled radioactively with dATP[α-35S] at the 3'-end using terminal deoxynucleotidyl transferase. Positive hybridization reactions were visualized by autoradiography in the normal pituitary gland with all of the probes. The clinically diagnosed pituitary adenomas (prolactinoma, acromegaly, Cushing's disease, FSH-secreting tumour) showed positive hybridization with the corresponding oligonucleotide probes. In some cases positive hybridization was also obtained with other probes, suggesting multihormone-producing character of the tumour cells. A microprolactinoma was found in a pituitary gland obtained from a patient without any known pituitary disorders. Examination of mRNAs for chromogranin A and B revealed that the normal pituitary gland contains a larger number of cells expressing chromogranin B and a lower number expressing chromogranin A and, moreover, the microprolactinoma lacked the expression of mRNA for chromogranin A but expressed that of chromogranin B.

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