S2367

On positively charged slides, 4 μm thick sections (FFPE) were dried for 20 min in 60°C. The slides were deparaffinised in Xylene and rehydrated in ethanol to water. Heat Induced Epitope Retrieval (HIER) was carried out by incubation of tissue sections in Target Retrieval Buffer pH 9.0 (WT1 and SIX2; S2367, DAKO), or pH 6.0 (CKAE1_2 and Ki67; S1699, DAKO) and 0,2% Triton X‐100 (T8787‐50ML, Sigma), in a Decloaking Chamber (NxGen, Biocare Medical, CA) for 20 min in 95°C. Endogenous peroxidase was blocked for 20 min with 1% H₂O₂ (95321‐100ML, Sigma–Aldrich) diluted in PBS pH 7.4 (A9201, 0010, Applichem). The sections were incubated with primary antibodies against WT1 (WT1‐RTU, clone 6F‐H2, DAKO), CKAE1_3 (MAB3412, Millipore), Ki67 (M7240, DAKO) and SIX2 (66347‐1‐lg). Except for WT1, which was prediluted, all primary antibodies were diluted in PBS containing 5% normal goat serum (005‐000‐001, Jackson Immuno Research). Immunostainings were visualised using BrightVision Poly‐HRP‐ Anti mouse RTU (DPVR55HRP, AH Diagnostics) for 30 min, followed by incubation with DAB (Liquid DAB+ Substrate Chromogen System, K3477, DAKO) for 5 min, and counterstained with Mayers Htx (01820, Histolab) for 30 s. All incubations were performed at room temperature and sections were washed three times with PBS after each incubation. Slides were mounted with Faramount Aqueous Mounting Medium (S3025, DAKO).

The expression patterns of WHSC1 in the OCCC samples and normal ovary specimens were confirmed by immunohistochemistry (IHC) (Additional file 1: Table S3). Briefly, we first performed deparaffinization and rehydration of the paraffin-embedded ovarian carcinoma specimens and normal ovarian tissue slides. Next, we microwaved the slides for 20 min with antigen retrieval buffer (pH 9; S2367, DAKO, Glostrup, Denmark). Anti-WHSC1 antibody (dilution: 1:200; ab75359, Abcam) was added to the tissue sections and incubated overnight at 4 °C. After washing with phosphate buffered saline (PBS), secondary antibody reaction was performed using substrate buffer (K5007, DAKO) and color development reaction with diaminobenzidine (DAB). We then stained the samples briefly with hematoxylin and then covered them with cover slips [18 ].

Mice testes for IHC and immunofluorescence staining were proceeded as described previously.^{33 (link), 34 (link)} The experiments were repeated three times. The antibodies used for IHC/immunofluorescence staining are listed in Supplementary Information. Specifically, antigen retrieval was carried out by heating slides in a pressure cooker (121 °C) for 10 min in a citrate buffer pH9.0 (S2367, DAKO, Glostrup, Denmark) for p53 staining and pH6.0 (S2369, DAKO) for other antibody staining. The peroxidase-conjugated secondary antibodies used were mouse/rabbit EnVisionC (DAKO) for HRP-immunostaining or anti-rabbit/mouse Alexa 488/568 IgG (Invitrogen, Carlsbad, CA, USA) for immunofluorescence staining. Images were captured with a Zeiss AxioImager upright microscope and Olympus FV1000 upright confocal microscope.^{34 (link)}

Paraffin-embedded sections of lung tumor tissue array (T8235732–5, BioChain) were processed in a microwave (90 °C) with antigen-retrieval solution (pH 9) (S2367; Dako), treated with a peroxidase-blocking reagent, and then treated with a protein-blocking reagent (K130, X0909; Dako). Tissue sections were incubated with rabbit anti-SMYD2 antibody (#9734 S; CST) followed by incubation with an HRP-conjugated secondary antibody (Dako). Immunoreactivity was visualized with a chromogenic substrate (Liquid DAB Chromogen; Dako). Finally, tissue specimens were stained with Mayer’s hematoxylin solution (Hematoxylin QS; Vector Laboratories) for 5 s to discriminate the nucleus from the cytoplasm. After the mice were sacrificed, the tumors and organs were collected and fixed in 10% formalin for 24 h. Then, the fixed tissues were sectioned and embedded in paraffin. Tissue Section (4 μm) were deparaffinized and then stained with hematoxylin and eosin (H&E), and Ki67 immunochemistry was performed according to a standard protocol. Images of the whole cross section were captured using an EasyScan slide scanner (Motic). Images were analyzed using Motic ImagePlus software (Motic).