Localization of cells expressing AChE mRNA in rat striatum using nonradioactive in situ hybridization.
A better understanding of the role of AChE in mammalian brain requires knowledge of the distribution of AChE synthesizing cells in this tissue. The aim of the present study is to test a nonradioactive approach for the localization of AChE mRNA positive cells in rat striatum. Nonradioactive in situ hybridization has not been used before for the localization of this mRNA in mammalian brain. In order to find optimal conditions for localization, we employed both RNA and oligonucleotide probes. We also examined various prehybridization protocols and approaches. The total number of cells in brain sections was determined by subsequent fluorescent staining of the nuclei. Optimal AChE mRNA localization was obtained with a digoxigenine-labeled RNA probe. We were not able to localize AChE mRNA with nonradioactively 3' end-labeled oligonucleotides. An acetylation step prior to hybridization was found to be essential for optimal signal/background ratios; high nonspecific staining was observed, if this step was omitted. In accordance with reports of other authors, who used radioactive in situ hybridization, we found very low percentages of AChE mRNA-positive cells in striatum, although this area exhibits very high AChE staining. In comparison to radioactive techniques, the nonradioactive approach avoids the risks of radioactivity, and is much less time consuming. In our experiments AChE mRNA localization in striatum was practically the same as that demonstrated previously by radioactive approaches.
Mis
K
K
Institute of Pathophysiology, School of Medicine, University of Ljubljana, Zaloska, Slovenia.
Zajc-Kreft
K
K
Grubic
Z
Z
D000110:Acetylcholinesterase / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D006690:Bisbenzimidazole; D001923:Brain Chemistry / Q000235:genetics; D003342:Corpus Striatum / Q000166:cytology* / Q000201:enzymology*; D005456:Fluorescent Dyes; D017403:In Situ Hybridization / Q000379:methods; D008297:Male; D015347:RNA Probes; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D012474:Salmon; D013194:Staining and Labeling
D005456:Fluorescent Dyes; D015347:RNA Probes; D012333:RNA, Messenger; D000110:Acetylcholinesterase; D006690:Bisbenzimidazole