Grino M, Zamora A J
Laboratoire de Neuroendocrinologie Expérimentale, INSERM U 297 and Interactions Cellulaires Neuroendocriniennes, UMR 6544 CNRS, Institut Jean Roche, UER de Médecine Nord, Marseille, France.
J Histochem Cytochem. 1998 Jun;46(6):753-9. doi: 10.1177/002215549804600608.
We present a specific and sensitive method for simultaneous detection of three mRNA species in individual neurons. The method relies on the use of riboprobes labeled with [35S]-UTP, digoxigenin-UTP, or biotin-UTP. The nonradioactive probes were sequentially revealed by incubation with anti-digoxigenin immunoglobulins or streptavidin conjugated to peroxidase, followed by the use of fluorochrome-labeled tyramides as peroxidase substrates. The radioactive probe was revealed by conventional autoradiography. There was no interaction among the different probes or the various detection systems. We demonstrate the use of this method by illustrating on laser scanning confocal microscopy the co-localization of the mRNAs coding for corticotropin-releasing factor (CRF), arginine vasopressin (AVP), or peptidylglycine alpha-amidating monooxygenase (PAM) in rat hypothalamic paraventricular nucleus (PVN) and its modulation by endogenous glucocorticoids. Our results suggest that this method could be used not only to study the regulation of the hypothalamo-pituitary-adrenal axis but also in various models in which mRNAs are present at low concentrations.
我们提出了一种用于同时检测单个神经元中三种mRNA种类的特异且灵敏的方法。该方法依赖于使用用[35S]-UTP、地高辛配基-UTP或生物素-UTP标记的核糖探针。非放射性探针通过与抗地高辛配基免疫球蛋白或与过氧化物酶偶联的链霉亲和素孵育依次显示，随后使用荧光染料标记的酪胺作为过氧化物酶底物。放射性探针通过传统放射自显影显示。不同探针或各种检测系统之间没有相互作用。我们通过在激光扫描共聚焦显微镜上展示编码促肾上腺皮质激素释放因子（CRF）、精氨酸加压素（AVP）或肽基甘氨酸α-酰胺化单加氧酶（PAM）的mRNA在大鼠下丘脑室旁核（PVN）中的共定位及其受内源性糖皮质激素的调节，来证明该方法的应用。我们的结果表明，该方法不仅可用于研究下丘脑-垂体-肾上腺轴的调节，还可用于mRNA以低浓度存在的各种模型。
