Areas with a high cellularity were selected for tissue microarrays. Immunohistochemical staining was performed as described previously [12 (link)]. E-cadherin (1:50 dilution; DAKO, Glostrup, Denmark; Catalogue No. M3612) and N-cadherin (1:500 dilution; Abcam, Cambridge, UK; Catalogue No. ab12221) antibodies were applied into a Bond-max autostainer system (Leica Microsystems, Bannockburn, IL, USA). Antigen retrieval was carried out using citrate buffer at pHÂ 6.0. Negative controls were prepared without using primary antibodies.
All immunostained slides were evaluated twice by two independent observers (NMG and LKH) with no knowledge of the clinical details. E- and N-cadherin immunohistochemistry showed cytoplastmic positivity in glioma cells and sometimes stained the cytoplasmic borders. The intensity of staining was initially classified into 4 grades: 0, no immunoreaction; 1, weak positivity; 2, moderate positivity; and 3, strong reactivity. With N-cadherin staining, cases of grades 0 and 1 positivity were grouped as a low-expression, and cases of grades 2 and 3 as a high-expression for statistical convenience. Two pathologists re-evaluated cases with discordant staining intensity together and made concessions for such cases.
All immunostained slides were evaluated twice by two independent observers (NMG and LKH) with no knowledge of the clinical details. E- and N-cadherin immunohistochemistry showed cytoplastmic positivity in glioma cells and sometimes stained the cytoplasmic borders. The intensity of staining was initially classified into 4 grades: 0, no immunoreaction; 1, weak positivity; 2, moderate positivity; and 3, strong reactivity. With N-cadherin staining, cases of grades 0 and 1 positivity were grouped as a low-expression, and cases of grades 2 and 3 as a high-expression for statistical convenience. Two pathologists re-evaluated cases with discordant staining intensity together and made concessions for such cases.
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