Signalstain kit

Tissue samples were fixed using 4% phosphate buffered formaldehyde and paraffin-embedded according to standard procedures. H&E staining was performed on 4 µm paraffin sections using standard protocols. Immunohistochemistry was performed on 3 µm paraffin-embedded tissue sections after deparaffinization and heat-induced epitope retrieval in citrate buffer by using the SignalStain Kit by Cell Signaling according to the manufacturer’s instructions. As primary antibody, anti-BRAF-V600 antibody (1:500, SAB5600047 SIGMA) (Supplementary Fig. 3S), anti-S6K (1:250 Anti-P70 S6 Kinase beta antibody, ab70963), anti-CD34 (1:600, Anti-CD34 antibody [EP373Y], ab81289), anti-p38 MAPK (1:1000, Anti-p38 antibody, ab197348) and anti-PTEN (1:1000, Anti-PTEN antibody, ab31392) was applied to the tissue and incubated overnight at 4 °C. The next day, after application of SignalStain® Boost IHC Reagent followed by SignalStain® DAB, counterstaining with Meyer’s haemalaun solution was performed. The samples were then mounted and analyzed with an Olympus microscope. Positive cells were counted (by ImageJ) in 6 high-fields (40x magnification) per slide and compared to the total number of cells in each field. From this data, the mean percentage of positive cells was calculated.

Tissue samples were fixed using 4% phosphate buffered formaldehyde and paraffin-embedded according to standard procedures. H&E staining was performed on 4 μm paraffin sections using standard protocols. Immunohistochemistry was applied using an autostainer (Dako) after heat-induced epitope retrieval in citrate buffer. IDH1 mutation was assessed by immunohistochemistry using an anti-IDH1-R132H antibody (1:20, Dianova). Immunohistochemistry was performed on 3 μm paraffin-embedded tissue sections after deparaffinization and heat-induced epitope retrieval in citrate buffer by using the SignalStain Kit by Cell Signaling according to the manufacturer’s instructions. As primary antibody, Anti-PD-L1-antibody (E1LRN by Cell Signaling) was applied to the tissue in a concentration of 1:200 and incubated overnight at 4°C. The next day, after application of SignalStain® Boost Solution and Secondary Antibody Solution, counterstaining with Meyer’s haemalaun solution was performed. The samples were then mounted and analyzed with an Olympus microscope. PD-L1 positive cells were counted in 6 high-fields (40x magnification) per slide and compared to the total number of cells in each field. From this data, the mean percentage of PD-L1 positive cells was calculated.