An in situ hybridization histochemistry method for the use of alkaline phosphatase-labeled oligonucleotide probes in small intestine.
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.
MRC group, Institute of Animal Physiology & Genetics Research, Babraham, Cambridge, United Kingdom.
H
H
Kiyama
P C
PC
Emson
D000469:Alkaline Phosphatase / Q000032:analysis* / Q000235:genetics; D000818:Animals; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D006651:Histocytochemistry; D006851:Hydrochloric Acid / Q000494:pharmacology; D007421:Intestine, Small / Q000737:chemistry* / Q000187:drug effects / Q000201:enzymology; D008297:Male; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D011142:Polyvinyl Alcohol / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains; D013004:Somatostatin / Q000032:analysis / Q000235:genetics
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D013004:Somatostatin; D011142:Polyvinyl Alcohol; D000469:Alkaline Phosphatase; D006851:Hydrochloric Acid