Mayer s hemalum

Tumors and lungs were zinc fixed and paraffin-embedded. Tumor sections were stained using the Opal staining system and analyzed with InForm software using the phenotyping tool according to the manufacturer's instructions (PerkinElmer, Rodgau, Germany). Tumor sections were stained with the following antibodies: cleaved caspase (Cell Signaling, Cambridge, U.K.); Ki67 (abcam, Cambridge, U.K.); hypoxia-inducible factor 1-alpha (HIF1α) (Novus); panCytokeratin (abcam); CD31 (BD); alpha smooth muscle actin (αSMA) (Sigma-Aldrich); spectral DAPI (PerkinElmer); neural/glial antigen 2 (NG2) (R&D systems, Minneapolis, USA). For metastases at least nine independent sections of each lung were stained with Mayer's hemalum (Merck, Darmstadt, Germany) and analyzed. Secondary antibody controls for each antibody species were routinely included (Supplementary Figure 1).

Slides were immune-labelled using the UltraVision LP Detection System (Thermo Scientific, Fremont, CA, USA) according to the manufacturer’s instructions. SR-BI (Novus Biologicals, Littleton, USA, 2 µg/mL, rabbit anti-human, polyclonal) or vWF (Dako, 0.725 µg/mL, immunoglobulin fraction, rabbit anti-human, polyclonal) were applied for 30 min at room temperature. After three washings in PBS-T, slides were incubated with primary antibody enhancer for 10 min, followed by HRP-Polymer for 15 min. The slides were washed again three times in PBS-T, and immune-labelling was visualized by a 5 min exposure to 3-amino-9-ethylcarbacole (ECA, all from UltraVision kit, Thermo Scientific). The slides were counterstained with Mayer’s hemalum (Merck, Darmstadt, Germany) and after washing in distilled water mounted with Kaiser’s glycerol gelatin (Merck).

After deparaffinization and rehydration, 3 to 5 μm thick tissue sections were placed for 10 min in 10 mM citrate buffer (pH 6.0) at 95°C for antigen unmasking, followed by treatment with 3% H₂O₂ for 10 min at room temperature to block endogenous peroxidase. Staining for STAT1 was performed with the Vectastain ABC Kit (Vector Laboratories, Burlingame, CA) using a 1:250 dilution of primary STAT1 rabbit monoclonal antibody (42H3, Cell Signaling) in SignalStain® Antibody Diluent (Cell Signaling) and overnight incubation at 4°C. Slides were treated with the chromogen diaminobenzidine and counterstained with Mayer’s Hemalum (Merck).

Adrenals were dissected, cleared of the fat tissue, and fixed overnight in 4% paraformaldehyde. Four-µm paraffin sections were processed for protein immunodetection as previously described (10 (link)). Briefly, antigen retrieval was performed in 10 mM Sodium Citrate pH 6, and incubation was conducted overnight using a mouse monoclonal anti-Disabled-2/p96 (Dab2; BD Transduction Laboratories, cat. no. 610464; RRID: AB_397837) and a rabbit polyclonal anti-Akr1b7 [kindly provided by Dr Pierre Val and Dr Antoine Martinez; RRID: AB_3075891 (34 (link))]. Indirect staining was performed using the goat anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody conjugated with Alexa Fluor™ 488 (from Thermo Fisher Scientific, cat. no. A11008; RRID: AB_143165), and a goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody conjugated with Alexa Fluor™ 647 (from Thermo Fisher Scientific, cat. no. A21235; RRID: AB_2535804). 4′,6-diamidino-2-phenylindole was used for counterstaining. Images were captured with a Nikon Eclipse Ti-E microscope. Hematoxylin and eosin staining was carried out on neighboring sections compared to the immunofluorescence experiment. For Oil Red O staining, mouse adrenals were snap frozen and 5-µm sections were processed in a mixed solution of Oil Red O and dextrin, followed by nuclear counterstaining with Mayer's hemalum (all products from Merck).

For immunohistochemical assessment of tumor microvascular density, tumor sections were incubated with a polyclonal rabbit anti-CD31 primary antibody (Abcam ab28364 1:50, Cambridge, United Kingdom) overnight. Further work-up of tissue samples was performed using the EnVision+ System HRP (AEC) (DAKO Diagnostika, Hamburg, Germany) according to the manufacturer´s instruction. Counterstaining was performed using Mayer´s hemalum (Merck Millipore, Darmstadt, Germany) and slides were covered with Kaiser´s Glycerin Gelatine (Merck Millipore, Darmstadt, Germany). Tumor microvessels were quantified as the average number of endothelial cells in 10 random fields at 200x magnification [32 (link)].

Ki-67-specific monoclonal rabbit anti-human antibody (SP6, Abcam ab16667 1:100, Cambridge, United Kingdom) was used to quantify tumor cell proliferation. The tissue was demasked in 1 x citrate buffer (pH = 6.0) using microwave irradiation at 600 W. After washing the slides in ddH₂O and TBS-Tween (0.05%) the multi-step kit EnVision+ System HRP (DAB) (DAKO Diagnostika, Hamburg, Germany) was used for the staining following the manufacturer’s instructions. Counterstaining was performed using Mayer´s hemalum (Merck Millipore, Darmstadt, Germany) and slides were covered with Kaiser´s Glycerin Gelatine (Merck Millipore, Darmstadt, Germany). Results were quantified as the average number of proliferating cells in 10 random fields at 200x magnification.