PITFALLS OF IMMUNOGOLD LABELING - ANALYSIS BY LIGHT-MICROSCOPY, TRANSMISSION ELECTRON-MICROSCOPY, AND PHOTOELECTRON MICROSCOPY
PITFALLS OF IMMUNOGOLD LABELING - ANALYSIS BY LIGHT-MICROSCOPY, TRANSMISSION ELECTRON-MICROSCOPY, AND PHOTOELECTRON MICROSCOPY
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DOI：
10.1177/35.8.2439584
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发表时间：
1987-08-01
影响因子：
3.2
通讯作者：
GRIFFITH, OH
中科院分区：
文献类型：
--
作者：
BIRRELL, GB;HEDBERG, KK;GRIFFITH, OH
免疫金法被广泛用于细胞抗原的定位、鉴定和区分。然而，有一些陷阱可能导致非特异性结合，特别是在细胞骨架研究中，由小金颗粒制备的金探针。我们提出了减少非特异性结合的建议清单，特别注意本研究中确定的两个问题。首先，我们发现制备胶体金颗粒的方法会影响非特异性结合的程度。其次，标准的bsa稳定的小金探针显然具有与细胞骨架制剂的蛋白质结合的暴露区域。利用光学显微镜、透射电子显微镜和银增强标本光电子显微镜对培养细胞的全贴装细胞骨架制备进行了研究。金探针由三种不同的还原剂（白磷、硼氢化钠和柠檬酸-单宁酸）还原HAuCl4产生的约5毫米颗粒制成。三种稳定的制剂均显示出显著的非特异性结合水平，其中柠檬酸-单宁酸的非特异性结合水平最高。通过在探针缓冲液中加入鱼明胶，用鱼明胶代替制备探针所用的BSA稳定剂，或采用预吸附法，这三种类型的探针在很大程度上解决了这个问题。这些技术的应用产生了清晰的免疫金标记模式和最小的非特异性背景。
The immunogold method is widely used to localize, identify, and distinguish cellular antigens. There are, however, some pitfalls that can lead to nonspecific binding, particularly in cytoskeletal studies with gold probes prepared from small gold particles. We present a list of suggestions for minimizing nonspecific binding, with particular attention to two problems identified in this study. First, we find that the method used to prepare the colloidal gold particles affects the degree of nonspecific binding. Second, the standard BSA-stabilised small gold probes evidently possess exposed regions that bind to the proteins of cytoskeletal preparations. This was investigated in whole-mount cytoskeletal preparations of cultured cells by use of light microscopy, transmission electron microscopy, and photoelectron microscopy of silver-enhanced specimens. Gold probes were made from approximately 5-mm particles generated by reduction of HAuCl4 with three different reducing agents: white phosphorus, sodium borohydride, and citrate-tannic acid. All three preparations stabilized in the conventional way showed significant levels of nonspecific binding, which was highest with citrate-tannic acid. This problem was largely solved with all three types of probes by including fish gelatin in the probe buffer, by substituting fish geltin for the BSA stabilizer used to prepare the probes, or by pre-adsorption methods. Application of these techniques resulted in clear immunogold labeling patterns with minimal nonspecific background.