Heavy Metal Enhancement Technique for Diaminobenzidine in Immunohistochemistry Enables Ultrastructural Observation by Low-vacuum Scanning Electron Microscopy
Heavy Metal Enhancement Technique for Diaminobenzidine in Immunohistochemistry Enables Ultrastructural Observation by Low-vacuum Scanning Electron Microscopy
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DOI：
10.1369/00221554221102996
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发表时间：
2022-05-25
影响因子：
3.2
通讯作者：
Shimizu, Akira
中科院分区：
文献类型：
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作者：
Arai, Yutaka;Takeuchi, Kazuhiro;Shimizu, Akira
低真空扫描电子显微镜（LV-SEM）是一种强大的工具，可以通过去除盖玻片，在更高的放大倍数下观察具有高碘酸银乌洛托品（PAM）染色的光学显微镜标本。然而，由于背散射电子图像不足，不适合使用3，3 '-二氨基联苯胺（DAB）进行免疫组织化学（IHC）观察。通过LV-SEM检查了IHC中DAB的传统重金属增强技术，（1）四氧化锇和铁，（2）钴，（3）乌洛托品银（Ag），（4）氯化金（Au），以及（5）Ag和Au（Ag + Au）。从Thy1.1肾小球肾炎大鼠肾脏组织标本染色与α-平滑肌肌动蛋白和DAB可视化增强这些增强方法。在光镜和左室扫描电镜下，我们发现，银，金，银+金的增强比其他的增强有更好的强度和对比度。在更高的放大倍数下，Ag + Gold增强显示高强度和低背景，尽管只有Ag或Gold增强具有非特异性背景。即使在通过LV-SEM观察之后，在重新安装盖玻片之后，样品的质量也得以保持。还证实Ag + Gold增强可用于使用临床人肾活检的IHC。这些结果表明，Ag +金在IHC中为LM和LV SEM观察提供了充分的增强。
Low-vacuum scanning electron microscopy (LV-SEM) is a powerful tool that allows to observe light microscopic specimens with periodic acid-silver methenamine (PAM) staining at a higher magnification, simply by removing the coverslip. However, it is not suitable for observation of immunohistochemistry (IHC) using 3,3 '-diaminobenzidine (DAB) due to insufficient backscattered electron image. Traditional heavy metal enhancement techniques for DAB in IHC, (1) osmium tetroxide and iron, (2) cobalt, (3) methenamine silver (Ag), (4) gold chloride (Gold), and (5) both Ag and Gold (Ag + Gold), were examined by LV-SEM. Tissue specimens from Thy1.1 glomerulonephritis rat kidney stained with alpha-smooth muscle actin and visualized with DAB were enhanced by each of these enhancement methods. We found, in light microscopic and LV-SEM, that the enhancement with Ag, Gold, or Ag + Gold had better intensity and contrast than others. At a higher magnification, Ag + Gold enhancement showed high intensity and low background, although only Ag or Gold enhancement had nonspecific background. Even after observation by LV-SEM, the quality of specimens was maintained after remounting the coverslip. It was also confirmed that Ag + Gold enhancement could be useful for IHC using clinical human renal biopsy. These findings indicate that Ag + Gold provided an adequate enhancement in IHC for both LM and LV SEM observation.