Comparison of horseradish peroxidase visualization methods: quantitative results and further technical specifics.
Comparison of horseradish peroxidase visualization methods: quantitative results and further technical specifics.
复制标题
辣根过氧化物酶可视化方法的比较：定量结果和进一步的技术细节。
DOI：
10.1177/29.8.7276535
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发表时间：
1981
期刊：
影响因子：
--
通讯作者：
D. Pfaff
中科院分区：
文献类型：
--
作者：
J. Morrell;L. Greenberger;D. Pfaff
通过对每种方法后发现的逆行标记神经元进行计数，定量比较了用于辣根过氧化物酶（HRP）神经组织化学证明的四种方法。HRP作为逆行标记物是一种重要的神经解剖学示踪方法，如果这些神经解剖学研究要完全显示传入神经元，其显示逆行标记神经元的最大灵敏度是很重要的。所比较的四种方法为二氨基联苯胺（DAB）法、对苯二胺和邻苯二酚的汉克-耶茨法、邻二氨基联苯胺法和四甲基联苯胺法（TMB）。TMB程序导致传入到HRP应用位点的神经元的更完整的地形，并证明所有传入细胞组中的神经元比其他三种程序中的任何一种都要多。使用TMB方法在小HRP应用的情况下尤其重要，这种大小对神经解剖学研究有用，而其他方法显示很少或没有逆行标记的神经元。如果神经元有小颗粒的反应产物（颜色随色素原而变化），描述了染色体形状，通常延伸到突起，并且细胞核清晰，则判断它们是HRP逆行标记的。此外，在邻二苯胺或TMB反应后，少数逆行标记的神经元的体细胞和突起特别充满了反应产物，使其看起来像来自高尔基体的神经元。对于灵敏的TMB反应，准确的H2O2浓度、新鲜配制的溶液、最小的处理后酒精暴露、反染色和清洁的玻璃器皿都是很重要的。
Four methods used for the neurohistochemical demonstration of horseradish peroxidase (HRP) were quantitatively compared by counting retrogradely labeled neurons found after each method was used. HRP used as a retrograde marker is an important neuroanatomical tracing method, and maximum sensitivity in its demonstration of retrogradely, labeled neurons is important if these neuroanatomical studies are to completely demonstrate afferent neurons. The four methods compared were a diaminobenzidine (DAB) procedure, a Hanker-Yates procedure using P-phenylenediamine and pyrocatechol, an o-dianisidine procedure, and a tetramethyl benzidine (TMB) procedure. The TMB procedure resulted in a more complete topography of neurons afferent to the HRP application site, and demonstrated many more neurons in all afferent cell groups that either of the three other procedures. Use of the TMB method was especially critical in the cases of small HRP applications, a size useful for neuroanatomical studies, where the other methods demonstrated very few or no retrogradely labeled neurons. Neurons were judged to be retrogradely HRP labeled if they had small granules of the reaction product (the color varying with the chromogen) describing the somal shape, usually extending into the processes, and a clear nucleus. In addition, after the o-dianisidine or the TMB reaction a small number of retrogradely labeled neurons had soma and processes especially well filled with reaction product, giving the appearance of neurons from Golgi preparations. For a sensitive TMB reaction giving good results, exact H2O2 concentration, freshly prepared solutions, minimal postreaction exposure to alcohol, counterstaining, and clean glassware were each found to be important.