Gocht A
Anatomisches Institut, Abteilung für Neuroanatomie, Universität Hamburg, BRD.
Acta Anat (Basel). 1992;145(4):327-39. doi: 10.1159/000147385.
The object of this study was to investigate the applicability of the acrylic resin 'LR White' to immunolabelling of various antigenic determinants in aldehydefixed rat CNS tissue. Antibodies were used, which worked well in paraffin sections and therefore were suitable to detect antigens resistant to complete dehydration and heat. Different LR White embedding protocols were employed in order to select the preparation conditions that adequately preserved both the antigenicity and fine structure. Specimens were completely dehydrated with up to 100% ethanol, which was followed by various infiltration times with LR White monomer. Polymerization of the resin was induced by heat, a chemical catalytic procedure (accelerator), or ultraviolet (UV) light. Paraffin, as well as semithin and ultrathin LR White sections were incubated with antibodies reacting to antigens located on the cell surface (stage-specific embryonic antigen-1; SSEA-1), within the plasma membrane (myelin basic protein), in the cytosol (HNK-1, S100 protein), in the cytoskeleton (GFAP, vimentin, neurofilament protein, INT-FIL), and in the extracellular matrix (laminin). All of the examined antigens were immunocytochemically detectable in paraffin-embedded material, while the carbohydrate moieties, HNK-1 and SSEA-1, were not immunoreactive in LR White sections. However, in cryostat sections processed for pre-embedding immunoelectron microscopy, the HNK-1 epitope and SSEA-1 were immunolabelled. Polymerization carried out under UV light led to better structural preservation of brain tissue than resin cured with heat or catalyst. The length of prior infiltration with monomer apparently had no effect on tissue preservation. Consequently, UV light-induced polymerization of LR White gives acceptable morphology of brain tissue. However, the use of this acrylic resin is restricted to the detection of some CNS antigens only.
本研究的目的是探讨丙烯酸树脂“LR White”在醛固定大鼠中枢神经系统组织中对各种抗原决定簇进行免疫标记的适用性。使用的抗体在石蜡切片中效果良好，因此适用于检测对完全脱水和加热有抗性的抗原。采用了不同的LR White包埋方案，以选择能充分保留抗原性和精细结构的制备条件。标本先用高达100%的乙醇完全脱水，然后用LR White单体进行不同时间的渗透。树脂的聚合通过加热、化学催化程序（促进剂）或紫外线（UV）光诱导。将石蜡切片以及LR White半薄切片和超薄切片与针对位于细胞表面（阶段特异性胚胎抗原-1；SSEA-1）、质膜内（髓鞘碱性蛋白）、胞质溶胶（HNK-1、S100蛋白）、细胞骨架（GFAP、波形蛋白、神经丝蛋白、INT-FIL）和细胞外基质（层粘连蛋白）中的抗原的抗体一起孵育。所有检测的抗原在石蜡包埋材料中均可通过免疫细胞化学检测到，而碳水化合物部分、HNK-1和SSEA-1在LR White切片中无免疫反应性。然而，在用于包埋前免疫电子显微镜的冰冻切片中，HNK-1表位和SSEA-1被免疫标记。与通过加热或催化剂固化的树脂相比，紫外线诱导的树脂聚合能更好地保存脑组织的结构。单体预先渗透的时间长短显然对组织保存没有影响。因此，紫外线诱导的LR White聚合能使脑组织具有可接受的形态。然而，这种丙烯酸树脂的使用仅限于仅检测某些中枢神经系统抗原。
