The Fluorescent Staining of Heparin in Mast Cells Using Berberine Sulfate: Compatibility with Paraformaldehyde oro-Phthalaldehyde Induced Fluorescence and Metachromasia

Abstract

The berberine sulfate technique of Enerbäck (1974) for the demonstration of heparin was applied to freeze-dried or routinely fixed paraffin embedded sections from various tissues. Sections from tissue which had been freeze-dried were deparaffinized, fixed in Carnoy Formula A for 15 minutes, rinsed in ethanol, hydrated, stained for 20 minutes in a 0.02% aqueous solution of berberine sulfate at a pH of 1, 2, 3, 4, 5 or 6 and rinsed in distilled water at a pH corresponding to the staining solutions. Carnoy Formula A or formalin-fixed tissues were routinely processed and sections were stained as above. Also, sections of freeze-dried tissues which had been deparaffinized and treated with paraformaldehyde or o-phthalaldehyde were hydrated to quench the fluorescence due to serotonin or histamine and restained with aqueous solutions of berberine sulfate (pH 1—6). In sections of tissue at a pH above 4, yellow fluorescence was produced by berberine sulfate in both cartilage and mast cells while in those treated at a pH of 4 and below this fluorescence was limited to heparin-containing mast cells. Sections which were stained with berberine sulfate later were counter-stained with azure A or methylene blue to verify that the cells containing fluorescence were metachromatic and with Alcian blue 8GX at pH 1-4 to demonstrate the specificity of berberine sulfate for heparin. The results of this study confirmed that the yellow fluorescence produced by berberine sulfate at a pH of 4 and below is specific for heparin in mast cells within sections of tissue. This study also indicated that staining with berberine sulfate is compatible with (1) paraffin embedded tissues which have been freeze-dried or prepared by routine histological methods, (2) histological sections which have been treated previously with paraformaldehyde for serotonin or o-phthalaldehyde for histamine fluorescence and (3) the subsequent counterstaining of tissue sections for metachromasia.