Highly sensitive radioactive in situ hybridization using full length hydrolyzed riboprobes to detect alpha 2 adrenoceptor subtype mRNAs in adult and developing rat brain.
The mRNA expression of highly homologous alpha 2 adrenoceptor subtypes was determined using a highly sensitive in situ hybridization protocol that allowed the detection of low abundance mRNA. Full-length 35S-labeled riboprobes specific for alpha 2A, alpha 2B and alpha 2C adrenoceptors were used for maximal sensitivity. The probes were hydrolyzed to an average length of 600 bp which, in combination with proteinase K digestion, resulted in optimal probe penetration in developmental and adult tissue. The expression intensity could be quantified and the ontogeny of receptor mRNA expression determined. At the same time receptor binding sites or functional proteins could be detected simultaneously in adjacent sections, because fresh frozen and post-fixed tissue was used.
Winzer-Serhan
U H
UH
Department of Pharmacology, College of Medicine, University of California, Irvine 92697, USA.
Broide
R S
RS
Chen
Y
Y
Leslie
F M
FM
D000818:Animals; D001345:Autoradiography / Q000379:methods; D001921:Brain / Q000196:embryology / Q000254:growth & development*; D001923:Brain Chemistry; D005629:Frozen Sections; D017403:In Situ Hybridization / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000235:genetics*; D015345:Oligonucleotide Probes; D020033:Protein Isoforms / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D018341:Receptors, Adrenergic, alpha-2 / Q000235:genetics*; D012680:Sensitivity and Specificity; D013048:Specimen Handling; D013462:Sulfur Radioisotopes / Q000032:analysis; D016707:Tissue Fixation