Publication protocol
Liver sections were prepared as follows. A small piece of liver tissue was rinsed in saline and placed in 4% phosphate-buffered formaldehyde and shaken gently. Samples were allowed to fix at 4 °C for 24 h, at which point they were embedded in paraffin blocks. Four 5 μm thick sections were cut using a cryostat (Microm, Francheville, France) and placed on glass slides (ChemMate, Dako). Slices were deparaffinized with two 15 min washes with xylene at room temperature, step-rehydrated with two 5 min washes in 100% ethanol and two 5 min washes in 95% ethanol, followed by one 10 min wash in 70% ethanol, and finally, two 5 min washes in doubly distilled water. Nonspecific sites were coated by incubating for 2 h in phosphate-buffered saline (PBS; 10 mmol·L−1 phosphate buffer, 150 mmol·L−1 NaCl, and 10 mmol·L−1 KCl (pH 7.4)) supplemented with 0.05% NaN3, 0.3% Triton X-100, 1% bovine serum albumin (Sigma), and 1% goat serum (Dako). Slides were rinsed by means of three 15 min washes in PBS and were incubated at 4–8 °C with rabbit polyclonal OAT antibody (1:75 dilution; Levillain et al. 2004) and mouse monoclonal glutamine synthetase antibody (dilution 1:75) (Chemicon International, Temecula, California, USA) in PBS supplemented with 0.05% NaN3, 0.3% Triton X-100, and 1% bovine serum albumin. Sections underwent three 15 min washes with the same buffer (without antibody). Alexa-conjugated antibodies were centrifuged at 10 000g for 2 min before use. For confocal microscopy, sections were incubated for 60 min at room temperature in the dark with PBS containing 0.5% gelatin and Alexa-488-conjugated affinity-purified goat anti-rabbit (dilution 1:300) and Alexa-633-conjugated affinity-purified goat anti-mouse (dilution 1:300; Jackson Laboratories, Inc.). Sections were washed for 2 h in the same buffer. Slices were counterstained for 3 min with 2 μg·mL−1 of Hoechst stain (Polysciences Inc.). Sections were washed for 2–6 h in PBS and mounted with Fluoprep (Biomerieux, France). Images were taken with a Leica confocal system TCS-SP2 (oil immersion objective, 63× magnification), using a filter set at 505–559 nm for Alexa 488 (OAT protein), 678–768 nm for Alexa 633 (glutamine synthetase protein), and 403–496 nm for the Hoechst stain. Photographs were analyzed using a Leica microscope (DMR, Germany) equipped with a color video camera (Kodak DC290 digital zoom) coupled to a computer (Mac OS9.2). All data were analyzed using Image J software. Pictures from confocal microscopy were analyzed and merged using Image J software and transferred to Adobe Photoshop CS3 extended version.
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