Sakanaka M, Magari S, Shibasaki T, Shinoda K, Kohno J
Department of Anatomy, Osaka Medical College, Japan.
J Histochem Cytochem. 1988 Sep;36(9):1091-6. doi: 10.1177/36.9.3136206.
A sensitive combination of horseradish peroxidase (HRP) tracing and immunohistochemistry was used by Rye et al. [J Histochem Cytochem (1984) 32:1145] in a search for the origins of neurotransmitter- and neuromodulator-containing nerve fibers in brain. In this combination, peroxidase as a marker in immunohistochemistry was thought to yield a homogeneous brown immunoreaction product of diaminobenzidine, different from the black granular reaction product of retrogradely transported HRP, which is visualized by the tetramethylbenzidine (TMB) reaction and subsequent stabilization. A neuron that exhibits both kinds of reaction products in its cytoplasm in sections subjected to combination staining is referred to as a double-labeled cell. With a combined HRP and corticotropin-releasing factor (CRF) immunoperoxidase-antiperoxidase (PAP) method, the first set of experiments showed "false" double-labeled cells in the pyramidal cell layer of rat cerebral cortex, but only rarely in the subcortical areas, possibly because of the use of one enzyme system in two different histochemical procedures. This limitation of the double-staining technique prompted us to demonstrate an alternate combination of HRP tracing and immunohistochemistry in the second set of experiments by employing two previously described independent enzyme systems: HRP as a retrograde tracer and beta-galactosidase as a marker for immunohistochemical demonstration of CRF. A homogeneous blue reaction product indicated immuno-beta-galactosidase staining, and a granular black or brown reaction product labeled retrogradely transported HRP in double-labeled cells in subcortical regions. Neither double labeling nor "false" double labeling was seen in pyramidal cells of cerebral cortex. These findings suggest that application of two independent enzyme systems in a combined HRP and immunohistochemical method may be useful for investigating in origins of peptidergic fibers in brain when the combination of HRP histochemistry and the PAP method appears to be inappropriate.
Rye等人[《组织化学与细胞化学杂志》（1984年）32:1145]使用辣根过氧化物酶（HRP）追踪和免疫组织化学的灵敏组合方法，探寻大脑中含有神经递质和神经调质的神经纤维的起源。在这种组合方法中，过氧化物酶作为免疫组织化学中的标记物，被认为会产生二氨基联苯胺的均匀棕色免疫反应产物，这与逆行运输的HRP的黑色颗粒反应产物不同，后者通过四甲基联苯胺（TMB）反应及随后的固定处理得以显现。在进行组合染色的切片中，细胞质内同时呈现这两种反应产物的神经元被称为双标记细胞。采用HRP与促肾上腺皮质激素释放因子（CRF）免疫过氧化物酶-抗过氧化物酶（PAP）联合方法进行的第一组实验显示，在大鼠大脑皮质的锥体细胞层出现了“假”双标记细胞，但在皮质下区域很少见，这可能是因为在两种不同的组织化学程序中使用了同一个酶系统。双染色技术的这一局限性促使我们在第二组实验中展示HRP追踪和免疫组织化学的另一种组合，即采用两种先前描述的独立酶系统：HRP作为逆行示踪剂，β-半乳糖苷酶作为CRF免疫组织化学示踪的标记物。均匀的蓝色反应产物表明是免疫β-半乳糖苷酶染色，而颗粒状黑色或棕色反应产物标记了皮质下区域双标记细胞中逆行运输的HRP。在大脑皮质的锥体细胞中既未观察到双标记，也未观察到“假”双标记。这些发现表明，当HRP组织化学与PAP方法的组合似乎不合适时，在联合HRP和免疫组织化学方法中应用两种独立酶系统，可能有助于研究大脑中肽能纤维的起源。
