Quantitative Autoradiography of Tyrosine Hydroxylase Immunoreactivity in the Rat Brain
Quantitative Autoradiography of Tyrosine Hydroxylase Immunoreactivity in the Rat Brain
复制标题
大鼠脑中酪氨酸羟化酶免疫反应性的定量放射自显影
作者：
R. Raisman‐Vozari;E. Hirsch;F. Javoy‐Agid;Cécile Vassort;M. Savasta;C. Feuerstein;J. Thibault;Y. Agid
采用快速、灵敏的放射免疫组织化学方法，在未固定的大鼠脑组织切片的离散区域定量测定酪氨酸羟化酶（TH）的水平。与TH单克隆抗体的免疫反应通过35S标记的二抗显示，从而允许对酶进行放射自显像检测。射线自显影是通过将组织切片与高灵敏度胶片相对置或浸入射线自显影乳剂中生成的。对抗体浓度、孵育时间、组织切片厚度和胶片曝光时间进行了详细分析，以确定产生与抗原量相关的线性放射性标记强度的最佳条件。通过计算机辅助图像分析评估区域水平抗原的定量。通过与组织切片平行处理构建的TH校准曲线，将脑区放射标记的放射自显影光密度转换为酶浓度。对标记的特异性、定量的有效性和重复性进行了研究。TH放射标记的分布与使用免疫荧光组织化学或在匀浆上测量TH酶活性所描述的分布相当。使用35S标记的抗体，可以在细胞水平上检测TH。
Levels of tyrosine hydroxylase (TH) were quantified in discrete areas of unfixed rat brain tissue sections using a rapid and sensitive radioimmunohistochemical method. The immunological reaction with the TH monoclonal antibody was revealed by a 35S‐labelled secondary antibody and thus permitted autoradiographic detection of the enzyme. Autoradiograms were generated by apposition of tissue sections to high‐sensitivity films or by dipping into autoradiographic emulsion. A detailed analysis of antibody concentration, incubation time, tissue section thickness, and exposure time of the film was undertaken to determine optimal conditions to produce a linear radiolabelling intensity with respect to the amount of antigen. Quantification of the antigen at regional levels was assessed by computer‐assisted image analysis. Autoradiographic optical density of radiolabelling in brain areas was converted to enzyme concentrations by interpolation with a constructed TH calibration curve processed in parallel with tissue sections. The specificity of the labelling and the validity and reproducibility of the quantification were investigated. The distribution of TH radiolabelling was comparable to that described using immunofluorescence histochemistry or measuring TH enzymatic activity on homogenates. Using a 35S‐labelled antibody, the detection of TH could be performed at the cellular level.
登录
查看更多内容
[3H]卡托普利对大鼠脑中血管紧张素转换酶的放射自显影可视化：定位至纹状体黑质通路。
DOI：
10.1073/pnas.81.5.1599
发表时间：
1984
影响因子：
11.1
作者：
Strittmatter,SM;Lo,MM;Javitch,JA;Snyder,SH
通讯作者：
Snyder,SH
碳水化合物在人绒毛膜促性腺激素中的作用：去糖基化使激素受体复合物和腺苷酸环化酶系统解偶联。
DOI：
10.1016/0006-291x(82)91880-0
发表时间：
1982
影响因子：
3.1
作者：
Thotakura,NR;Bahl,OP
通讯作者：
Bahl,OP
DOI：
--
发表时间：
1984-07
期刊：
The Journal of pharmacology and experimental therapeutics
影响因子：
--
作者：
E. Richelson;A. Nelson
通讯作者：
E. Richelson;A. Nelson
影响因子：
5.8
作者：
F. Sulser;J. Vetulani;P. Mobley
通讯作者：
F. Sulser;J. Vetulani;P. Mobley
重复用去甲基丙咪嗪和尼亚酰胺治疗后，大鼠松果体β-肾上腺素能受体连接的腺苷酸环化酶系统的敏感性降低。
影响因子：
3.6
作者：
Moyer,JA;Greenberg,LH;Frazer,A;Weiss,B
通讯作者：
Weiss,B