Binding of ten FITC-labelled lectins (Vector Laboratories, Burlingame, CA, USA) was tested to investigate the composition and distribution of oligosaccharidic chains in mucins (Table 1 and references therein29-38 (link)). Sections were incubated for 1 h at room temperature with the lectin solution in HEPES and subsequently rinsed in the same buffer and mounted in Fluoromount (Sigma-Aldrich). Each experiment was repeated twice on each sample.
Controls for lectin labelling were performed by i) substitution of lectin with HEPES alone; ii) incubation in lectin with inhibitory sugar added (0.2-0.5 M); iii) binding to the egg envelope of the toads Bombina pachypus and Bufo bufo, the mucins of which are demonstrated to be labelled by the tested lectins.39 (link),40 (link)Lectin-binding was observed in epifluorescence under 495 nm light emission. Photos were taken by a Nikon DMX 1200 camera coupled with a Nikon Eclipse 600 photomicroscope (Nikon Instruments SpA, Calenzano, FI, Italy).
Controls for lectin labelling were performed by i) substitution of lectin with HEPES alone; ii) incubation in lectin with inhibitory sugar added (0.2-0.5 M); iii) binding to the egg envelope of the toads Bombina pachypus and Bufo bufo, the mucins of which are demonstrated to be labelled by the tested lectins.39 (link),40 (link)Lectin-binding was observed in epifluorescence under 495 nm light emission. Photos were taken by a Nikon DMX 1200 camera coupled with a Nikon Eclipse 600 photomicroscope (Nikon Instruments SpA, Calenzano, FI, Italy).