Jirikowski G F, Ramalho-Ortigao J F, Kesse K W, Bloom F E
Department of Neuropharmacology, Scripps Clinic and Research Foundation, La Jolla, CA 92037.
Histochemistry. 1990;94(2):187-90. doi: 10.1007/BF02440186.
We recently described a nonradioactive method for in situ hybridization with 5-bromo-2-deoxyuridine (BrdU) labelled oligonucleotide probes. An antibody to BrdU and immunocytochemistry were used in order to detect the hybridization signal. We have now applied this method to semithin Epon sections, in order to hybridize consecutive sections through single cells with different probes and to stain them with antibodies to neuropeptides. It could be shown that Epon embedding reserves mRNA well. In the present study we used a BrdU labelled synthetic oligonucleotide probe complementary to a fragment of the vasopressin precursor and an antibody to Arg-vasopressin. Vasopressin mRNA was demonstrable in a fraction of the vasopressin immunoreactive neurons in the magnocellular nuclei. In addition some of the magnocellular neurons showed either hybridization or vasopressin immunostaining only, perhaps indicating different stages of synthetic and secretory activity. The method described seems to be a valuable tool for studying synthetic activity in peptidergic neurons on a single cell level. The method might also have potential for in situ hybridization on the electron-microscopical level.
我们最近描述了一种用于与5-溴-2-脱氧尿苷（BrdU）标记的寡核苷酸探针进行原位杂交的非放射性方法。使用抗BrdU抗体和免疫细胞化学来检测杂交信号。我们现在已将此方法应用于半薄Epon切片，以便用不同的探针杂交单个细胞的连续切片，并用神经肽抗体对其进行染色。可以证明，Epon包埋能很好地保存mRNA。在本研究中，我们使用了与加压素前体片段互补的BrdU标记的合成寡核苷酸探针和抗精氨酸加压素抗体。在大细胞核对加压素免疫反应性神经元的一部分中可检测到加压素mRNA。此外，一些大细胞神经元仅显示杂交或加压素免疫染色，这可能表明合成和分泌活动的不同阶段。所描述的方法似乎是在单细胞水平上研究肽能神经元合成活性的一种有价值的工具。该方法在电子显微镜水平的原位杂交中也可能具有潜力。
