HRP和AP呈现束缚-浸水应激大鼠NTS儿茶酚胺能神经元Fos表达的效果比较

Effect of HRP and AP Labeling on Fos Expression in Catecholamine Neurons of the NTS of Rats During Restraint Water-immersion Stress

用辣根过氧化物酶(HRP)单酶先后标记Fos和酪氨酸羟化酶(TH),用二氨基联苯胺(DAB)增强剂和DAB分别呈色;以及同时用碱性磷酸酶(AP)和HRP双酶分别标记TH和Fos,再分别用5-溴-4-氯-3-吲哚磷酸盐(BCIP)和氯化硝基四氮唑蓝(NBT)对TH和DAB对Fos呈色,比较免疫双标时两种不同酶标记显色方法对研究大鼠孤束核(NTS)内儿茶酚胺能神经元在束缚-浸水应激中作用效果的影响.两种显色方法的免疫阳性神经元都清晰易辩,结果应激组NTS内Fos,TH及Fos/TH双标阳性神经元数目均显著增加.但HRP单酶双底物显色系统耗时长、非特异性着色强,在抗体种属来源不同时可优先考虑使用HRP/AP双酶双底物显色.

A dual immunohistochemical technique is used to detect the effect of horse radish peroxidase(HRP) and alkaline phosphatase(AP) labeling on Fos expression in catecholaminergic neurons of the neucleus of solitary tract(NTS) of rats during restraint water-immersion stress(RWIS).One method is that horse radish peroxidase(HRP) labeled Fos and TH in turn,and then using diaminobenzidine hydrochloride(DAB),intensified with 0.05% cobalt chloride and 0.05% nickel ammonium sulfate,to visualize Fos-immunoreactive neurons(Fos-IR),and DAB to TH-immunoreactive neurons(TH-IR).The other method is that HRP labeled Fos and alkaline phosphatase(AP) labeled TH neurons,and then using DAB and 5-bromo-4-chloro-3-indolylphosphate(BCIP)/nitroblue tetrazolium(NBT) to visualize Fos-IR and TH-IR respectively.Fos-IR nuclei,TH-IR,Fos and TH double labeled neurons evidently increase in stressed rats.The two methods all fit to reveal the phenotypic nature of the activated neurons.But using HRP labeling Fos and TH respectively would cost much more time and lead to a nonspecific immunoreactivity,and if the antibodies come from different species in origin,HRP/AP is a priority.