在同一神经元内同时显示Fos－蛋白、酪氨酸羟化酶和HRP逆行标记的三重标记技术  

STUDIES ON METHODS FOR ELECTROFUSION OF MOUSE 2-CELL EMBRYOS

作　　者：饶志仁[1] 陈良为[1] 晋光荣[1] 韩智安 董元祥[1] 熊抗辉[1] 施际武[1] 

机构地区：[1]第四军医大学解剖学教研室,南京铁道医学院解剖学教研室

出　　处：《解剖学报》1994年第2期219-223,T020,共5页Acta Anatomica Sinica

基　　金：国家自然科学基金

摘　　要：本文介绍了一种能在同一神经元内同时显示ＨＲＰ逆行标记以及Ｆｏｓ和某类神经递质或相关酶（例如ＴＨ）的三重标记技术。首先将ＨＲＰ注入脑内某一核团（如中央杏仁核），动物存活４８ｈ后，再向胃内导入１％福尔马林２ｍｌ，以造成对胃的伤害性刺激，２ｈ后将动物处死。脑的切片（如本文的延髓切片）先用四甲基联苯胺－钨酸钠（ＴＭＢ－ＳＴ）法进行ＮＲＰ呈色反应，后用二氨基联苯胺（ＤＡＢ）和氯化钻作加强；然后按ＡＢＣ法进行Ｆｏｓ免疫组织化学反应，继之按ＰＡＰ法进行ＴＨ免疫组织化学反应。光镜下在延髓切片观察到７种标记神经元，即ＴＨ－、ＨＲＰ－或Ｆｏｓ－单标记神经元，ＴＨ和ＨＲＰ－、ＴＨ和Ｆｏｓ－或ＨＲＰ和Ｆｏｓ的双标记神经元以及ＴＨ和ＨＲＰ和Ｆｏｓ的三标记神经元。该方法为研究同时显示中枢神经元的功能活动状态、传出投射及所含神经递质提供了一个强有力的工具。The parameters of electrofusion of 2-cell mouse embryos were optimized for application as a model for nuclear transplantation.There was considerable lysis of embryos with M2 as the medium for fusion;however,100% fusion(n=58)was obtained with a single 0.31kV/cm,1280μsec pulse.With mannitol and sucrose solution as the medium, however,a wide range of field strengths(0.31-1.41kV/cm for 0.26 mol/L sucrose solution and 0.31-2.04kV/cm for 0.3 mol/L mannitol solution)and durations of the electrical pulse(10-1280 μsec)resulted in high rates of fusion(often 100%).Likewise,osmolarity of sucrose and mannitol solutions did not affect the rate of fusion using a 0.47kV/cm pulse.With a field strength of 2.04kV/cm,however,the proportion of embryos that fused in mannitol solution increased(P<0.05)and the proportion of lysed embryos,decreased(P<0.05)as osmolarity increased.Both fused(162/642,25%)and control embryos(32/72,44%)continued to develop in culture for 48 hours ,and thereafter began to compact.Fused embryos were only at the 4-cell stage by this time,while control embryos were at the 8-cell stage.Optimal pulse durations are plotted for field strengths between 0.31 and 1.41kV/cm with 0.26 mol/L sucrose as fusion medium.

关 键 词：Fos-蛋白 免疫组织化学 神经元 

分 类 号：R322-33[医药卫生—人体解剖和组织胚胎学]