Lea P, Lee L M, Shi Q W, Takahashi M, Youn W, Jackowski G
Spectral Diagnostics Inc., Toronto, Ontario, Canada.
Scanning. 1996 Jun;18(4):259-68. doi: 10.1002/sca.1996.4950180401.
Myoglobin and myosin light chain 1 (MLC1) are intracellular human cardiac marker proteins which are released as a consequence of ischemia. Human cardiomyocytes were isolated from fresh biopsies and also maintained for several passages in cell culture. The cardiomyocytes were fixed in 100% methanol at -20 degrees C, and labeled. The immunolocalization of intracellular antigen by fluorescence conjugated imaging was compared with scanning electron microscopy (SEM) backscatter electron (BSE) imaging of gold conjugated antibody. Ultra-violet light microscopy showed the intracellular distribution of both proteins to be mainly in the nuclear envelope, the cytoplasm immediately surrounding the nucleus and along portions of the cell membrane. To confirm this observed distribution of myoglobin and MLC1, labeling was repeated with antimyoglobin and anti-MLC1 monoclonal antibodies conjugated to colloidal gold particles. The advantage of colloidal gold labeling is that the intracellular antigen-antibody complexes may be more precisely located because of the significant improvement in resolution provided by BSE imaging in the SEM. BSE imaging confirmed the presence and subsarcolemma localization of myoglobin in cardiomyocytes directly isolated from fresh biopsies. The distribution of colloidal gold-conjugated antibodies did not coincide with the intracellular distribution of the two proteins in the cardiomyocytes grown in cell culture as indicated by immunofluorescence. A relatively random, intracellular gold particle distribution was confirmed by x-ray microanalysis. BSE imaging resulted in consistent auto-backscatter labeling patterns very similar to the labeling patterns obtained with immunofluorescent labeling. X-ray microanalysis confirmed that these auto-backscatter labeling patterns were formed by concentrations of intracellular phosphate. Sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) and subsequent Western blotting indicated that myoglobin and MLC1 were no longer present in detectable quantities in these cells after several passages. Polymerase chain reaction (PCR) amplification of mRNA for human myoglobin and cardiac MLC1 confirmed the absence of their transcripts. Electrophoretic analysis of proteins in cardiomyocytes grown in cell culture confirmed an increasing presence of alkaline phosphatase. Staining of this enzyme with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium showed that alkaline phosphatase was distributed in the same intracellular pattern as the fluorescence conjugated anti-body and the phosphatase auto-backscatter. These results indicate that high-resolution backscatter SEM imaging may be used as necessary control to confirm fluorescence light microscope intracellular labeling of antigens.
肌红蛋白和肌球蛋白轻链1（MLC1）是细胞内的人类心脏标志物蛋白，它们会因缺血而释放。人类心肌细胞从新鲜活检组织中分离出来，并在细胞培养中传代培养了几代。心肌细胞在-20℃下用100%甲醇固定，然后进行标记。通过荧光共轭成像对细胞内抗原的免疫定位与金共轭抗体的扫描电子显微镜（SEM）背散射电子（BSE）成像进行了比较。紫外光显微镜显示这两种蛋白在细胞内的分布主要在核膜、紧邻细胞核的细胞质以及部分细胞膜上。为了证实观察到的肌红蛋白和MLC1的这种分布，用与胶体金颗粒共轭的抗肌红蛋白和抗MLC1单克隆抗体重复进行标记。胶体金标记的优点在于，由于SEM中的BSE成像提供的分辨率显著提高，细胞内抗原-抗体复合物可能定位得更精确。BSE成像证实了直接从新鲜活检组织中分离的心肌细胞中肌红蛋白的存在及其在肌膜下的定位。如免疫荧光所示，但胶体金共轭抗体的分布与在细胞培养中生长的心肌细胞中这两种蛋白的细胞内分布不一致。通过X射线微分析证实了细胞内金颗粒分布相对随机。BSE成像产生的一致的自动背散射标记模式与免疫荧光标记获得的标记模式非常相似。X射线微分析证实这些自动背散射标记模式是由细胞内磷酸盐浓度形成的。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）及随后的蛋白质印迹分析表明，传代培养几代后，这些细胞中不再能检测到肌红蛋白和MLC1。对人类肌红蛋白和心脏MLC1的mRNA进行聚合酶链反应（PCR）扩增，证实其转录本不存在。对在细胞培养中生长的心肌细胞中的蛋白质进行电泳分析，证实碱性磷酸酶的存在越来越多。用5-溴-4-氯-3-吲哚磷酸和硝基蓝四唑对这种酶进行染色，结果显示碱性磷酸酶的细胞内分布模式与荧光共轭抗体和磷酸酶自动背散射的模式相同。这些结果表明，高分辨率背散射SEM成像可作为必要的对照，以确认荧光显微镜下细胞内抗原的标记。
