Labeled polymer hrp anti rabbit

5um thick deparaffinized tissue sections were subjected to antigen retrieval using pressure cooker for 5 minutes in 1mM EDTA buffer. Endogenous peroxidase was quenched with 3% hydrogen peroxide for 10 minutes. Blocked sections were labeled with rabbit polyclonal anti-SCD1 antibody (abcam; 0.5mg/ml, 100ul) at the concentration of 6ug/ml overnight at 4°C. Anti-rabbit HRP-labeled polymer (DAKO) was used as a secondary antibody. Color detection was performed by liquid DAB+ substrate chromogen system (DAKO). Primary antibody was omitted in control reaction. Immunohistochemical scoring of SCD1 expression was performed regarding area and grade. The criteria were defined as follows: Over 70% of the tumor cells stained positively was scored as high (3 scores); less than 70% of the tumor cells stained positively was scored as medium (2 scores); less than 30% of the tumor cells were positively was scored as low (1 score). No color was scored as 0; light yellow staining was scored as 1; yellow staining was scored as 2; dark yellow staining was scored as 3. The final score was calculated by area scores and grade scores.

IHC analysis was performed on 4 μm FFPE tumor sections prepared from blocks corresponding to the same “exploratory” and “validation” cohort samples submitted for WES and cancer gene panel sequencing, respectively. Of the 79 aGCTs analyzed by genomic sequencing (WES or T200.1 cancer gene panel sequencing), one sample from the “exploratory” cohort did not have FFPE material available and one sample from the “validation” cohort had only a poor quality FFPE block that was not suitable for IHC; thus, 77 out of the 79 tumors in the combined cohort were able to be analyzed by IHC.
All slides were de-paraffinized in xylene and rehydrated. A heat-induced antigen retrieval step was employed using a citrate buffer at pH 6.0 (LabVision). Slides were incubated in peroxidase blocking solution (Dako) for 10 min at room temperature. All antibodies were then applied for 60 min at room temperature after dilution in an antibody diluent. KMT2D antibody was diluted in serum-free diluent (Dako). Slides were washed, and then incubated with anti-rabbit HRP-labeled polymer (Dako). Staining was visualized with diaminobenzidine (DAB) reagent (LabVision) and slides were counterstained with hematoxylin to visualize nuclei. KMT2D expression was detected using a previously validated^{20 (link)} rabbit polyclonal antibody directed against the KMT2D C terminus (HPA035977, Sigma-Aldrich) (1:500 dilution).