Optimization of Alcian blue pH1.0 histo-staining protocols to match mass spectrometric quantification of sulfomucins and circumvent false positive results due to sialomucins.

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).