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. 2021 Dec 7;11:23554. doi: 10.1038/s41598-021-02923-7

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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IHC analysis to study the expression of NT5E and MME in controls and GBC cases. (A) Representative IHC images showing the expression of NT5E and MME in controls and GBC cases. IHC was performed on formalin-fixed paraffin-embedded (FFPE) tissue microarray (TMA) and individual tissue sections. An in-house TMA block was constructed using the FFPE blocks and included 6 controls (6 GSD cases) and 14 GBC cases (2 early stage and 12 advanced stage). Each TMA block consisted of tissue cores of 2 mm diameter and 4 µm sections were cut from the TMA block for carrying out IHC. Individual tissue sections (FFPE) of 33 GBC (11 early stage and 22 advanced stage) and 17 controls (10 GSD and 7 XGC cases) were also for IHC analysis. IHC results showed positive expression for NT5E in 53.84% of early and 50% of advanced stage GBC cases while MME showed positive expression in 23.07% of early and 23.52% of advanced stage GBC cases. (B) The statistical analysis showed a significant difference between cases and controls for NT5E and MME for both early stage and advanced stage GBC. The scale bar is shown as red line. IHC scoring is shown in “Methodology” section “Immunohistochemistry analysis”.