In situ hybridization with digoxigenin labeled oligonucleotide probes: detection of CAMK-II gene expression in primary cultures of cerebral endothelial cells.
A better understanding of the regulation of gene expression under physiological and experimental conditions is one of the most important goals of today's neurobiology. In order to accomplish this task a number of in-situ hybridization methods have been elaborated. In the past few years the use of nonradioactive procedures have gained more and more space among these efforts. One of the most promising methods in this field is the application of digoxigenin-labeled probes, which have been used successfully in several laboratories. Here we present a rapid method providing good spatial resolution and low background labeling for the detection of messenger RNAs in various cell culture systems using digoxigenin-labeled probes. By using oligonucleotides complementary to the alpha and beta subunit of the calcium/calmodulin dependent protein kinase II (CAMK-II) we were able to demonstrate the presence of this enzyme in cultured cerebral endothelial cells, an enzyme that plays an important role in mediating the effects of extracellular signals.
Krizbai
I
I
Laboratory of Molecular Neurobiology, Institute of Biophysics, Biological Research Center, Szeged Hungary.
Deli
M
M
Lengyel
I
I
Maderspach
K
K
Pákáski
M
M
Joó
F
F
Wolff
J R
JR
D000818:Animals; D001921:Brain / Q000201:enzymology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics*; D002478:Cells, Cultured; D004076:Digoxigenin / Q000378:metabolism*; D004727:Endothelium / Q000166:cytology / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006651:Histocytochemistry; D017403:In Situ Hybridization; D015345:Oligonucleotide Probes / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004076:Digoxigenin