Abstract

Sulfomucins are in some body locations and species a normal occurrence, whereas in other situations, are a sign of pathology. Sulfomucin content on histological sections and isolated material is frequently analyzed with Alcian blue staining at pH 1.0. However, since the stain detects the charge, a high density of other charged molecules, such as sialic acids, has potential to impede specificity. Here, we compared the outcome from four staining protocols with the level of sulfation determined by liquid chromatography–tandem mass spectrometric analysis on samples from various tissues with variable sulfation and sialylation levels. We found that a protocol we designed, including rinsing with MetOH and 0.5 M NaCl buffer at pH 1.0, eliminates the false positive staining of tissues outperforming commonly recommended solutions. In tissues with low-to-moderately sulfated mucins (e.g. human stomach and salmonid epithelia), this method enables accurate relative quantification (e.g. sulfate scoring comparisons between healthy and diseased tissues), whereas the range of the method is not suitable for comparisons between tissues with high sulfomucin content (e.g. pig stomach and colon).

中文翻译：

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优化 Alcian blue pH 1.0 组织染色方案以匹配磺胺粘蛋白的质谱定量并避免由于唾液粘蛋白​​引起的假阳性结果

摘要

磺胺粘蛋白在某些身体部位和物种中是正常出现的，而在其他情况下，则是病理的征兆。组织切片和分离材料上的磺胺粘蛋白含量经常在 pH 1.0 下用阿新蓝染色进行分析。然而，由于染色剂检测到电荷，高密度的其他带电分子，如唾液酸，有可能阻碍特异性。在这里，我们将四种染色方案的结果与通过液相色谱 - 串联质谱分析确定的硫酸化水平对来自不同硫酸化和唾液酸化水平的各种组织的样本进行了比较。我们发现，我们设计的方案，包括在 pH 1.0 下用 MetOH 和 0.5 M NaCl 缓冲液冲洗，消除了优于通常推荐的解决方案的组织假阳性染色。