Lectin histochemistry (LHC) and immunohistochemistry (IHC), which exhibit the composition and localisation of sugar residues and proteins in cell membranes, respectively, are typically used individually.
Utilizing these two strategies, we beforehand demonstrated that malignant transformation of urothelial cells ends in the alterations of protein glycosylation and diminished expression of urothelium-specific integral membrane proteins uroplakins (UPs).
Nevertheless, the correlation between these adjustments was not studied but. To judge this correlation, we developed modern methodology, which we named mixed lectin- and immuno- histochemistry (CLIH).
We used human biopsies of 6 regular urothelia and 9 papillary urothelial carcinomas, i.e. three papillary urothelial neoplasms of low malignant potential (PUNLMP), three non-invasive papillary urothelial carcinomas of low grade (pTa, l.g.), and three invasive papillary urothelial carcinomas of excessive grade (pT1, h.g.).
We examined 5 completely different protocols (numbered 1-5) of CLIH on paraffin and cryo-semithin sections and in contrast them with LHC and IHC carried out individually. Moreover, we carried out western and lectin blotting with antibodies towards UPs and lectins Amaranthus caudatus agglutinin (ACA), Datura stramonium agglutinin (DSA), and jacalin, respectively.
We confirmed that incubation with main antibodies first, adopted by the combination of secondary antibodies and lectins is essentially the most environment friendly CLIH methodology (protocol quantity 5). Moreover, 300 nm thick cryo-semithin sections enabled higher decision of co-localisation between sugar residues and proteins than 5 µm thick paraffin sections.
Within the regular urothelium, CLIH confirmed co-localisation of lectins ACA and jacalin with UPs within the apical plasma membrane (PM) of superficial umbrella cells. In papillary urothelial carcinomas, all three lectins (ACA, DSA and jacalin) labelled areas of apical PM, the place they often co-localised with UPs.
Western and lectin blotting confirmed the variations between regular urothelium and papillary urothelial carcinomas. Our outcomes present that CLIH, when used with varied units of lectins and antigens, is a helpful, fast, and dependable methodology that could possibly be utilized for primary cell biology analysis in addition to detailed subtyping of human urothelial carcinomas.