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

• Sulfomucins are in some body-locations and species a normal occurrence, whereas in other situations a sign of pathology. Sulfomucin content on histological sections and isolated material is frequently analyzed with Alcian blue staining at pH1.0. 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 spectrometry analysis (MS) on samples from various tissues with variable sulfation and sialylation levels. We found that a protocol we designed, including rinsing with MetOH and 0.5M NaCl buffer at pH1.0 eliminates 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).

Ämnesord

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinsk bioteknologi -- Annan medicinsk bioteknologi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Medical Biotechnology -- Other Medical Biotechnology (hsv//eng)

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinsk bioteknologi -- Medicinsk bioteknologi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Medical Biotechnology -- Medical Biotechnology (hsv//eng)

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine (hsv//eng)

Nyckelord

Alcian blue

O-glycosylation

mass spectrometry

mucin

sulfation

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