Wouterlood F G
Department of Anatomy, Faculty of Medicine, Vrije Universiteit, Amsterdam, The Netherlands.
Histochemistry. 1988;89(5):421-8. doi: 10.1007/BF00492597.
In order to associate specific fiber projections in the central nervous system with specific target neurons, procedures were developed in which the anterograde neuroanatomical tracing technique utilizing Phaseolus vulgaris-leucoagglutinin (PHA-L) is combined with immunocytochemistry of three (different) neuronal markers: gamma-amino butyric acid, choline acetyltransferase, and serotonin. A double, indirect, peroxidase-antiperoxidase staining method is used on free-floating brain sections. The primary antiserum against the PHA-L (first primary antiserum) is mixed with the primary antiserum against the neuronal marker (second primary antiserum). These primary antisera are raised in different animal species. Following the incubation in the cocktail of two secondary antisera. The transported PHA-L is then visualized by incubation in a peroxidase-antiperoxidase complex and subsequent reaction with nickel-enhanced diaminobenzidine/H2O2 (blue reaction product in PHA-L-labeled neurons and fibers). Incubation is continued with peroxidase-antiperoxidase antibodies raised in the animal species in which the second primary antiserum is developed, and the staining is completed by treatment with diaminobenzidine/H2O2 (brown reaction product in target neurons). The present results suggest that PHA-L-tracing can be combined with immunocytochemistry of a variety of target neuron-related antigens.
为了将中枢神经系统中的特定纤维投射与特定靶神经元联系起来，人们开发了一些方法，即将利用菜豆白细胞凝集素（PHA-L）的顺行神经解剖示踪技术与三种（不同的）神经元标志物（γ-氨基丁酸、胆碱乙酰转移酶和5-羟色胺）的免疫细胞化学相结合。对游离脑切片采用双重间接过氧化物酶-抗过氧化物酶染色法。将抗PHA-L的第一抗血清（第一抗血清）与抗神经元标志物的第一抗血清（第二抗血清）混合。这些第一抗血清是在不同动物物种中产生的。在两种第二抗血清的混合物中孵育后，通过在过氧化物酶-抗过氧化物酶复合物中孵育并随后与镍增强的二氨基联苯胺/H2O2反应（PHA-L标记的神经元和纤维中产生蓝色反应产物）来使运输的PHA-L可视化。继续用在产生第二抗血清的动物物种中产生的过氧化物酶-抗过氧化物酶抗体进行孵育，并通过用二氨基联苯胺/H2O2处理（靶神经元中产生棕色反应产物）来完成染色。目前的结果表明，PHA-L示踪可与多种靶神经元相关抗原的免疫细胞化学相结合。
