Bond polymer refine red detection kit

Four µm sections were cut from FFPE tissue blocks and were heated for 30 min at 60°C. IDO1 staining was executed on an automated and validated Leica Bond-Max stainer (Leica Microsystems, Wetzlar, Germany). In brief, after dewaxing heat-induced epitope retrieval was performed in a high pH EDTA buffer (Bond Epitope Retrieval 2, Leica Microsystems) for 30 min. Slides were incubated with the primary IDO antibody (clone 10.1, Merck, Darmstadt, Germany) at a 1:50 dilution for 15 min and the antibody was detected via BOND Polymer Refine Red Detection kit (Leica Microsystems). Slides were then counterstained with hematoxylin.
IDO1 expression was scored in immune cells in the paracortex and in the sinuses of the lymph node, excluding the germinal centers. In the paracortex, the intensity of IDO1 staining was scored semiquantitatively using a four-tiered grading system (Figure 2A): no expression (0), weak expression (1+), moderate expression (2+) or strong expression (3+). For further analysis, IDO1 expression in the paracortex was dichotomized into an IDO1-low group (0 and 1+) and an IDO1-high group (2+ and 3+). In the sinuses, IDO1 staining was scored as ‘low’ or ‘high’ (Figure 2B).

Immunohistochemical studies were performed automatically in the Bond Max equipment (Leica Microsystems Inc.). Antigen retrieval steps were performed in Bond-Epitope Retrieval Solution 1 (Leica Microsystems, Germany) with TAGLN antibody (1:500, Abcam ab1416) at 100 °C. Detection was carried out with Bond Polymer Refine Red Detection kit (Leica Microsystems, DS9390). Stained slides were dehydrated and covered with mounting medium (Dako, CS70330) and cover-slips. Digital images of the slides were evaluated using the H-score method [84 (link)]. Statistical differences between groups were calculated with Mann–Whitney test while correlations were tested with Spearman correlation, in GraphPad Prism 6.0.