Hermanson O, Ericson H, Sanchez-Watts G, Watts A G, Blomqvist A
Department of Cell Biology, Faculty of Health Sciences, University of Linköping, Sweden.
J Histochem Cytochem. 1994 Jun;42(6):827-31. doi: 10.1177/42.6.7514627.
We describe a protocol for simultaneous light microscopic visualization of a neuron's efferent projections and its expression of mRNA. We have combined immunohistochemical visualization of the retrograde marker cholera toxin subunit B (CTb) with autoradiographic visualization of 35S-labeled cRNA probes. Injections of CTb were made into rat brain. Immunoreactivity for CTb was demonstrated by modification of the peroxidase-anti-peroxidase immunohistochemical technique, with DAB and nickel ammonium sulfate or cobalt acetate as chromogen. On the same sections, in situ hybridization was performed with a 35S-labeled RNA probe complementary to preproenkephalin mRNA or tyrosine hydroxylase mRNA. Many double-labeled neurons were detected. These neurons contained peroxidase reaction product and were covered by an accumulation of silver grains in the overlaying emulsion layer. The present method has several advantages over double-labeling methods using the combination of fluorescent tracers and oligonucleotide probes. Both reaction products are permanent and can be visualized simultaneously by light microscopy. Furthermore, both CTb and cRNA probes are very sensitive markers. In addition, the sections can be counterstained.
我们描述了一种用于同时在光学显微镜下观察神经元传出投射及其mRNA表达的方法。我们将逆行标记物霍乱毒素B亚基（CTb）的免疫组织化学可视化与35S标记的cRNA探针的放射自显影可视化相结合。将CTb注射到大鼠脑中。通过改良的过氧化物酶-抗过氧化物酶免疫组织化学技术，以二氨基联苯胺（DAB）和硫酸镍铵或醋酸钴作为显色剂，显示CTb的免疫反应性。在同一切片上，用与前脑啡肽原mRNA或酪氨酸羟化酶mRNA互补的35S标记RNA探针进行原位杂交。检测到许多双标记神经元。这些神经元含有过氧化物酶反应产物，并被覆盖在其上的乳剂层中的银颗粒积累所覆盖。与使用荧光示踪剂和寡核苷酸探针组合的双标记方法相比，本方法具有几个优点。两种反应产物都是永久性的，并且可以通过光学显微镜同时观察到。此外，CTb和cRNA探针都是非常敏感的标记物。另外，切片可以进行复染。
