Lectin histochemistry (LHC) and immunohistochemistry (IHC), which demonstrate the composition and localisation of sugar residues and proteins in cell membranes, respectively, are generally used separately. Using these two methods, we previously demonstrated that malignant transformation of urothelial cells results in the alterations of protein glycosylation and reduced expression of urothelium-specific integral membrane proteins uroplakins (UPs). However, the correlation between these changes was not studied yet. To evaluate this correlation, we developed innovative method, which we named combined lectin- and immuno- histochemistry (CLIH). We used human biopsies of 6 normal urothelia and 9 papillary urothelial carcinomas, i.e. 3 papillary urothelial neoplasms of low malignant potential (PUNLMP), 3 non-invasive papillary urothelial carcinomas of low grade (pTa, l.g.), and 3 invasive papillary urothelial carcinomas of high grade (pT1, h.g.). We tested five different protocols (numbered 1-5) of CLIH on paraffin and cryo-semithin sections and compared them with LHC and IHC performed separately. Additionally, we carried out western and lectin blotting with antibodies against UPs and lectins Amaranthus caudatus agglutinin (ACA), Datura stramonium agglutinin (DSA), and jacalin, respectively. We showed that incubation with primary antibodies first, followed by the mixture of secondary antibodies and lectins is the most efficient CLIH method (protocol number 5). Additionally, 300 nm thick cryo-semithin sections enabled better resolution of co-localisation between sugar residues and proteins than 5 µm thick paraffin sections. In the normal urothelium, CLIH showed co-localisation of lectins ACA and jacalin with UPs in the apical plasma membrane (PM) of superficial umbrella cells. In papillary urothelial carcinomas, all three lectins (ACA, DSA and jacalin) labelled regions of apical PM, where they occasionally co-localised with UPs. Western and lectin blotting confirmed the differences between normal urothelium and papillary urothelial carcinomas. Our results show that CLIH, when used with various sets of lectins and antigens, is a useful, quick, and reliable method that could be applied for basic cell biology research as well as detailed subtyping of human urothelial carcinomas.

通常分别分别使用凝集素组织化学（LHC）和免疫组织化学（IHC）来证明糖残基和蛋白质在细胞膜中的组成和定位。使用这两种方法，我们先前证明了尿路上皮细胞的恶性转化导致蛋白质糖基化的改变和尿路上皮特异性整合膜蛋白尿激酶（UPs）的表达降低。但是，尚未研究这些变化之间的相关性。为了评估这种相关性，我们开发了创新的方法，我们将其称为凝集素和免疫组织化学联合法（CLIH）。我们使用了6例正常尿路上皮和9例乳头状尿路上皮癌的人体活检，即。3例低恶性潜力的乳头状尿路上皮肿瘤（PUNLMP），3例低度的非浸润性乳头状尿路上皮癌（pTa，lg）和3例高度的浸润性乳头状尿路上皮癌（pT1，hg）。我们在石蜡切片和冷冻半胱氨酸切片上测试了五个不同的CLIH方案（编号1-5），并将它们与分别进行的LHC和IHC进行了比较。此外，我们用抗UPs和凝集素Amaranthus caudatus凝集素（ACA），曼陀罗的抗体进行了Western和凝集素印迹凝集素（DSA）和贾卡林。我们表明，首先与一抗一起孵育，然后与二抗和凝集素的混合物孵育是最有效的CLIH方法（协议编号5）。此外，与5 µm厚的石蜡切片相比，300 nm厚的冷冻半胱氨酸切片能够更好地解析糖残基和蛋白质之间的共定位。在正常的尿道上皮细胞中，CLIH显示凝集素ACA和jacalin与UPs在表层伞状细胞的顶质膜（PM）中共定位。在乳头状尿路上皮癌中，所有三种凝集素（ACA，DSA和jacalin）都标记了顶端PM的区域，有时与UPs共定位。Western和凝集素印迹证实正常尿路上皮癌和乳头状尿路上皮癌之间的差异。我们的结果表明CLIH，