Gross D K, De Boni U
Department of Physiology, University of Toronto, Ontario, Canada.
J Histochem Cytochem. 1990 Jun;38(6):775-84. doi: 10.1177/38.6.2139886.
We report a novel technique that combines high-resolution scanning electron microscopy (SEM) of intracellular structures with backscattered electron imaging (BEI) of colloidal gold-labeled intracellular ligands. Murine dorsal root ganglia were immersion-fixed, freeze-cleaved, labeled with gold complexes, and critical point-dried. Specimens were carbon-coated and viewed by BEI. They were then minimally sputter-coated with gold and previously identified cells relocated by secondary electron imaging (SEI). This permitted increased resolution of intracellular detail while gold particles remained detectable by BEI. Incubation with RNAse-gold and DNAse-gold complexes resulted in specific labeling of cytoplasm and nucleus, respectively. Immunolabeling of neurofilament (NF) and small nuclear ribonucleoproteins (snRNP) resulted in selective labeling of intracellular antigens. Nonspecific binding was abolished by use of 1% skin milk. Specifically, incubation with monoclonal anti-NF68 resulted in labeling of cytoplasm in 66% of neurons, notably of the large cells known to contain large amounts of NF. Satellite cells, which lack NF, showed low levels of background label. Human autoimmune anti-Sm serum recognizes snRNP particles, with the exception of the nucleolar U3 snRNP. Labeling with this serum resulted in specific labeling of 92% of nuclei, with only background labeling over nucleoli and cytoplasm. The results show that it is feasible to employ high-resolution SEM in conjunction with colloidal gold labeling to localize intracellular ligands in situ.
我们报告了一种新技术，该技术将细胞内结构的高分辨率扫描电子显微镜（SEM）与胶体金标记的细胞内配体的背散射电子成像（BEI）相结合。将小鼠背根神经节进行浸入固定、冷冻断裂、用金复合物标记，然后临界点干燥。标本进行碳涂层并通过BEI观察。然后用金进行最小限度的溅射涂层，并通过二次电子成像（SEI）重新定位先前识别的细胞。这在金颗粒仍可通过BEI检测到的同时，提高了细胞内细节的分辨率。用核糖核酸酶-金和脱氧核糖核酸酶-金复合物孵育分别导致细胞质和细胞核的特异性标记。神经丝（NF）和小核核糖核蛋白（snRNP）的免疫标记导致细胞内抗原的选择性标记。使用1%的皮肤乳消除了非特异性结合。具体而言，用单克隆抗NF68孵育导致66%的神经元细胞质被标记，特别是那些已知含有大量NF的大细胞。缺乏NF的卫星细胞显示出低水平的背景标记。人自身免疫抗Sm血清识别snRNP颗粒，但核仁U3 snRNP除外。用这种血清标记导致92%的细胞核特异性标记，仅在核仁和细胞质上有背景标记。结果表明，结合胶体金标记使用高分辨率SEM原位定位细胞内配体是可行的。
