Benchmark ultra machine

Tissue sections were processed sequentially: (1) Deparaffinisation at 72 °C, with Cell Conditioning using CC1 at 100 °C for 72 min; (2) Incubation with primary antibody for 1 h; and (3) Counterstain with Haematoxylin for 8 min and Bluing Reagent for 4 min. Standard protocol for IHC was followed using the Ventana Benchmark Ultra Machine. Individual primary antibodies were applied at the following concentrations: MLH1 (Ventana) - 1.4 μg/ml; MSH2 (Cell Marque) - 4.67 μg/ml; PMS2 (Cell Marque) - 2.19 μg/ml; MSH6 (Cell Marque) - 0.101 μg/ml. Visualisation of immuno-reactivity was performed using the Optiview Dab IHC detection Kit.

First, obtained surgical samples were fixed in formalin and paraffin blocks were prepared, using standard procedure. Sections (2–3 µm thick) were cut from these blocks and mounted on positively charged slides. The primary antibodies used for assay were GFAP and Ki67 (Ventana, ready to use). Antigen retrieval was performed on board (Ventana Benchmark Ultra machine), using high pH and detection by Ventana Ultraview (for GFAP) and Venta Optiview (for Ki67) detection kit. IDH1 was performed by Dianova, 1:50 and antigen retrieval was performed in PT link (Dako), high pH with detection by FLEX Dako in Dako autostainer. Finally, slides were counterstained with haematoxylin.