Faramount aqueous mounting medium

Paraffin‐embedded 5‐μm sections from tissue microarray slides of multiple liver diseases (LV1201; US Biomax, Rockville, MD, USA) were deparaffinized with xylene and rehydrated with alcohol. The slides were stained with anti‐DGAT1 and anti‐integrin β1 antibodies [diluted 1:100 in 1% bovine serum albumin (BSA) and 0.3% Triton X‐100]. After washing with PBS, the slides were incubated with FITC‐conjugated goat anti‐rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as the secondary antibody. Nuclei were counterstained with Hoechst 33342 (Invitrogen) for 15 min. After washing with PBS, the slides were mounted in Faramount aqueous mounting medium (DakoCytomation, Carpinteria, CA, USA) and the fluorescence signal was visualized using confocal or two‐photon microscopy (Carl Zeiss, Gottingen, Germany).

Tissue microarray (TMA) blocks comprising tissue cores (1 mm) with a sufficient proportion of tumor cells obtained from formalin-fixed paraffin-embedded (FFPE) tumor tissue blocks were used for immunohistochemistry (IHC) analysis of ERα, AR, glucocorticoid receptor (GR), PR, ERβ, PD-1, and PD-L1 (5-μm-thick sections cut using a rotary microtome), whereas whole tissue sections were used for the IHC analysis of CD4+, CD8+, CD3+, and FoxP3+. The sections were deparaffinized and rehydrated with graded ethanol. Then, the sections were treated with 3% H₂O₂ solution in methanol for 30 min to suppress endogenous peroxidase activity. Thereafter, heat-induced antigen retrieval was performed by incubating the sections in a target retrieval buffer at pH 6.0 (Dako, Carpinteria, CA, USA) using a steam pressure cooker (Pascal; Dako) for 20 min, and the slides were stained with the primary antibodies listed in Supplementary Table S5 with Autostainer Plus (Dako) for 1 h at room temperature. Then, EnVision+ Dual Link System-HRP (Dako) and DAB+ (3,3′-diaminobenzidine; Dako) were used for the visualization of antigen-antibody reactions. After dehydrating and counterstaining with hematoxylin, the slides were mounted in Faramount Aqueous Mounting Medium (Dako). Proper positive and negative controls were included.

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).