Roth J
J Histochem Cytochem. 1983 Aug;31(8):987-99. doi: 10.1177/31.8.6190857.
A method is described for the electron microscopic detection of lectin-binding sites in different cellular compartments and extracellular structures that uses thin sections from resin-embedded tissues. Various lectins (Ricinus communis lectin I and II, peanut lectin, Lotus tetragonolobus lectin, Ulex europeus lectin I, Lens culinaris lectin, Helix pomatia lectin, and soybean lectin) were bound to particles of colloidal gold and used for direct staining of thin sections or glycoprotein--gold complexes were prepared and applied in an indirect technique (concanavalin A and horseradish peroxidase--gold complex; wheat germ lectin and ovomucoid--gold complex). The details for preparation of such complexes from 14 nm gold particles are reported. The conditions of tissue processing that gave satisfactory staining results and good fine structure preservation were mild aldehyde fixation without osmification and low temperature embedding with the hydrophilic resin Lowicryl K4M. None of the so-called etching procedures was necessary prior to labeling of Lowicryl K4M thin sections. Examples of the use of this approach for detection of glycoconjugates in the rough endoplasmic reticulum, Golgi apparatus, and mucin of intestinal goblet cells as well as plasma membrane and various intracellular structures of absorptive intestinal and renal tubular cells are shown. A comparison is made with preembedding staining results on Concanavalin A-binding site localization in rat liver which shows that problems of penetration common in such a technique are circumvented by the postembedding approach described here. Concanavalin A-binding sites were not only consistently found in nuclear envelope, rough and smooth endoplasmic reticulum, plasma membranes, and collagen fibers, but also in mitochondria, glycogen, ribosomes, and nucleus. These data and those of a previous investigation (Roth J, Cytochem 31:547, 1983) prove the applicability of this cytochemical technique for postembedding localization of glycoconjugates by light and electron microscopy.
本文描述了一种利用树脂包埋组织的超薄切片，在电子显微镜下检测不同细胞区室和细胞外结构中凝集素结合位点的方法。将各种凝集素（蓖麻凝集素I和II、花生凝集素、四角豆凝集素、欧洲荆豆凝集素I、菜豆凝集素、蜗牛凝集素和大豆凝集素）与胶体金颗粒结合，用于超薄切片的直接染色，或者制备糖蛋白-金复合物并采用间接技术应用（伴刀豆球蛋白A和辣根过氧化物酶-金复合物；麦胚凝集素和卵类粘蛋白-金复合物）。报告了用14nm金颗粒制备此类复合物的详细方法。能获得满意染色结果并良好保存精细结构的组织处理条件为：采用温和的醛固定而不进行锇化处理，并用亲水性树脂Lowicryl K4M进行低温包埋。在标记Lowicryl K4M超薄切片之前，无需进行任何所谓的蚀刻程序。展示了该方法用于检测粗面内质网、高尔基体、肠杯状细胞粘蛋白以及吸收性肠细胞和肾小管细胞的质膜及各种细胞内结构中糖缀合物的实例。将其与大鼠肝脏中伴刀豆球蛋白A结合位点定位的包埋前染色结果进行了比较，结果表明此处所述的包埋后方法避免了该技术中常见的穿透问题。伴刀豆球蛋白A结合位点不仅始终存在于核膜、粗面和滑面内质网、质膜及胶原纤维中，还存在于线粒体、糖原、核糖体和细胞核中。这些数据以及先前一项研究（罗斯J，细胞化学31:547，1983）的数据证明了这种细胞化学技术在光镜和电镜下对糖缀合物进行包埋后定位的适用性。
