GLYCAN DETECTION BY LECTIN HISTOCHEMISTRY

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Methods: Lectin histochemistry was carried out using biotinylated lectin-streptavidin peroxidase system. Briefly, sections were deparaffinized in xylene, hydrated in downgraded ethanol and distilled water, respectively. The endogenous peroxidase was eliminated by treating sections with 3% H2O2 in water for 30 min. Sections were then washed in TBS (20 mM Tris-HCL, pH 7.4, containing 0.15 M NaCl, 10mM CaCl2 and 1mM MnCl2). Non-specific staining was blocked by treating slides with blocking solution (supplied by the kit) for 1 h. Biotinylated lectins (Table 1, Vector, USA) 10 ¿g /ml in TBS were applied to the sections and incubated for 1 h at room temperature. After washing with TBS for 30 min, the sections were incubated with Vectastain ABC reagent (Vector, USA) for 30 min. Color development using diaminobenzidine (DAB) reagent was done after washing with TBS for 30 min. Sections were then counterstained with Mayer's hematoxylin, dehydrated, cleared in xylene, and mounted with DePex mounting medium.