Free bovine serum albumin bsa

Tumor and metastasis samples obtained from animals were embedded in paraffin wax and cut in 10 µm sections for hematoxylin–eosin staining. For lung immunohistochemistry, 5 μm sections were cut, dewaxed, and rehydrated. Sections were treated with absolute methanol/3% H₂O₂ for 15 min to quench endogenous pseudoperoxidase activity, rinsed in distilled water and then in Tris–HCl buffer. After blocking non-specific binding for 20 min with horse serum (Vecton, Carpinteria, CA, USA), tissue sections were incubated with anti-S-Nitroso-Cysteine. The antibody was reconstituted and diluted in 0.01 M PBS containing 1% IgG-free bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) and 0.02% NaN₃. Bound antibodies were detected using the R.T.U VECTASTAIN Detection Kit (Vector Laboratories) and peroxidase was visualized with Liquid-3,3ʹdiaminobenzidine (DAB) + Substrate Chromogen System (DAKO). Slides were counterstained with hematoxylin–eosin and mounted with mounting medium (DAKO). Cells were visualized using a light microscope (Zeiss).

For all placental perfusion experiments Dulbecco´s modified eagle medium (DMEM, phenol red free, Gibco, UK) was used. DMEM was mixed with Earl´s buffer (6.8 g/L NaCl, 0.4 g/L KCl, 0.14 g/L NaH₂PO₄, 0.2 g/L MgSO₄•7 H₂O, 0.2 g/L CaCl₂, 2.2 g/L NaHCO₃, all Merck, Darmstadt, Germany) in a 3:1 medium buffer ratio containing amoxicillin (250 mg/L, Sigma-Aldrich, Steinheim, Germany), dextran FP40 (10 g/L, Serva, Heidelberg, Germany), 2 g/L glucose (Merck, Darmstadt, Germany), and essential fatty acid free bovine serum albumin (BSA) (5 g/L, Sigma-Aldrich, Seinheim, Germany).
Perfusion medium containing ¹³C-labeled free fatty acid mix (¹³C-FFA mix) was prepared under a stream of argon to prevent fatty acid oxidation. The albumin to FFA molar ratio was 0.77, which is in the physiological range in the systemic circulation of a pregnant women [9 (link), 23 (link)]. Preparation details and FFA final concentrations are shown in Supplementary Table 1.