Tammi R H, Hyyryläinen A M, Maibach H I, Tammi M I
Department of Anatomy, University of Kuopio, Finland.
Histochemistry. 1987;87(3):243-50. doi: 10.1007/BF00492417.
Fixation and staining procedures were developed for the electron microscopic demonstration of glycosaminoglycans (GAGs) in human epidermis. En bloc staining with cuprolinic blue (CB), ruthenium red (RR) and tannic acid (TA) in the primary fixative were applied for the localization of the GAGs. Removal of the epidermal basal lamina and underlying dermis was a prerequisite for stain penetration. In CB-fixed specimens 50 nm long, rod-like granules were found attached to keratinocyte cell surfaces, while the RR- and TA-fixed specimens contained round granules (luminal diameter 10 and 30 nm, respectively). The stainability of the CB-positive granules in the presence of 0.3 mol/l MgCl2 indicated that they contained sulphated GAGs. Prefixation digestions of epidermal sheets with chondroitinase ABC. Streptomyces hyaluronidase, and heparitinase showed that the RR-positive granules also contained sulphated GAGs, mostly heparan sulphate. The granules visualized with TA on keratinocytes were susceptible to heparitinase treatment, but the abundance of TA-staining suggested that TA also stained structures other than heparan sulphate. The EM data was in accordance with the 35SO4 labelling experiments showing that heparan sulphate was the major sulphated GAG synthesized in epidermis, whereas chondroitin/dermatan sulphates comprised about one fifth of the total activity incorporated. The distributions of the CB-, RR- and TA-positive granules on cell surfaces were similar. The morphology of the proteoglycan granules was probably determined by the extent of the GAG-chain collapse following binding to each of the dyes.
已开发出用于在电子显微镜下显示人表皮中糖胺聚糖（GAGs）的固定和染色程序。在初次固定剂中用亚铜灵蓝（CB）、钌红（RR）和单宁酸（TA）进行整体染色，以定位GAGs。去除表皮基底膜和下方的真皮是染色剂渗透的先决条件。在CB固定的50 nm长的标本中，发现杆状颗粒附着在角质形成细胞表面，而RR和TA固定的标本中含有圆形颗粒（管腔直径分别为10和30 nm）。在0.3 mol/l MgCl2存在下CB阳性颗粒的可染色性表明它们含有硫酸化GAGs。用软骨素酶ABC、透明质酸酶和硫酸乙酰肝素酶对表皮片进行预固定消化表明，RR阳性颗粒也含有硫酸化GAGs，主要是硫酸乙酰肝素。用TA在角质形成细胞上显示的颗粒易受硫酸乙酰肝素酶处理的影响，但TA染色的丰富程度表明TA也对硫酸乙酰肝素以外的结构进行染色。电子显微镜数据与35SO4标记实验一致，表明硫酸乙酰肝素是表皮中合成的主要硫酸化GAG，而硫酸软骨素/硫酸皮肤素约占总掺入活性的五分之一。CB、RR和TA阳性颗粒在细胞表面的分布相似。蛋白聚糖颗粒的形态可能由与每种染料结合后GAG链塌陷的程度决定。
