Merighi A, Polak J M, Fumagalli G, Theodosis D T
Department of Histochemistry, Royal Postgraduate Medical School, London, UK.
J Histochem Cytochem. 1989 Apr;37(4):529-40. doi: 10.1177/37.4.2564404.
Several immunogold techniques were used to determine the ultrastructural localization of calcitonin gene-related peptide (CGRP), tachykinin, somatostatin, and gamma-amino-butyric acid (GABA) immunoreactivity in the dorsal horn of rat spinal cord. The immunocytochemical reactions were carried out directly on ultrathin sections from non-osmicated frozen tissue, non-osmicated low temperature-embedded (Lowicryl K4M) tissue, and osmicated epoxy-embedded material. Preservation of ultrastructural morphology and immuno-labeling efficiency were compared. Morphology of subcellular organelles was relatively good in ultra-thin frozen sections, which showed the highest immunoreactivity. However, only very small samples of tissue could be examined. Although there was relatively good immunolabeling in the Lowicryl K4M-embedded tissue, the ultrastructure of the neuropil, and particularly that of synapses, was poorly maintained. In contrast, the osmicated epoxy-embedded material offered optimal morphological preservation together with accurate subcellular localization of all antigens under study. The latter approach thus enabled clear visualization of CGRP, tachykinin, and somatostatin immunoreactivity restricted to large dense-cored vesicles (90-150 nm diameter) in many axonal and synaptic profiles in the superficial layers of the dorsal horn. CGRP- and tachykinin-positive profiles were also present in the tract of Lissauer. GABA immunoreactivity was present mainly in axons and terminals, and less frequently in somatic and dendritic profiles. In terminals, which often formed symmetrical synapses on immunonegative dendritic profiles, it was associated with small (30-60 nm diameter) clear vesicles and mitochondria. Double immunolabeling was possible on all preparations, but the osmicated, epoxy-embedded material clearly showed co-localization of peptides, especially of CGRP and tachykinins, within the same dense-cored vesicles in axonal fibers and/or terminals. On the other hand, peptide and GABA immunoreactivity were consistently seen in different nerve profiles. In a few cases, GABAnergic terminals were seen to synapse on tachykinin-positive fibers.
采用几种免疫金技术来确定大鼠脊髓背角中降钙素基因相关肽（CGRP）、速激肽、生长抑素和γ-氨基丁酸（GABA）免疫反应性的超微结构定位。免疫细胞化学反应直接在未用锇酸处理的冷冻组织超薄切片、未用锇酸处理的低温包埋（Lowicryl K4M）组织以及用锇酸处理的环氧树脂包埋材料上进行。比较了超微结构形态的保存情况和免疫标记效率。超薄冷冻切片中亚细胞器的形态相对较好，其免疫反应性最高。然而，只能检查非常小的组织样本。尽管在Lowicryl K4M包埋组织中有相对较好的免疫标记，但神经毡的超微结构，尤其是突触的超微结构，保存较差。相比之下，用锇酸处理的环氧树脂包埋材料在提供最佳形态保存的同时，还能对所有研究抗原进行准确的亚细胞定位。因此，后一种方法能够清晰地观察到背角浅层许多轴突和突触轮廓中，CGRP、速激肽和生长抑素免疫反应性局限于直径90 - 150 nm的大型致密核心囊泡中。CGRP和速激肽阳性轮廓也存在于Lissauer束中。GABA免疫反应性主要存在于轴突和终末，较少见于胞体和树突轮廓。在终末，其常与免疫阴性的树突轮廓形成对称突触，与直径30 - 60 nm的小清亮囊泡和线粒体相关。所有制剂都可以进行双重免疫标记，但用锇酸处理的环氧树脂包埋材料清楚地显示了肽类，尤其是CGRP和速激肽，在轴突纤维和/或终末的同一致密核心囊泡内的共定位。另一方面，肽和GABA免疫反应性始终见于不同的神经轮廓。在少数情况下，可见GABA能终末与速激肽阳性纤维形成突触。
