Mounting media

After animal sacrifice, the brain was soaked in formalin after dehydration and 3-μm-thick microtome TMA slides were embedded in paraffin wax. The slides were individually immunostained with anti-ANP antibodies, employing the Ultra Vision LP Detection System (Vector Laboratories, Waltham, MA, USA). The embedded tissue was kept overnight at 60 °C for the pull-out embedded paraffin. After overnight rehydration with serious xylene and graded ethanol, “H₂O₂” blocking buffer was used to block peroxidation on embedded tissue and then washed with “ddH₂O”. The slides were treated with citrate buffer by heating with a microwave for the required time interval after cooling at room temperature. These slides were incubated with primary antibody for 2 h (1:100) after blocking with blocking buffer. Thereafter, the slides were incubated with the universal secondary antibodies for 30 min before staining with hematoxylin, and the slides were stained with chromogen for 5 min. Finally, slides were counterstained with hematoxylin, dehydrated by a series of graded ethanol to xylene washes, and fixed on coverslips with mounting media (Sigma Chemical, Saint Louis, MO, USA). Mounted tissue sections were viewed under a microscope (magnification 200×).

Immunohistochemistry was carried out on free floating sections after patching to confirm recording locations. Immediately after patch clamp recordings, VTA sections were fixed using 4% paraformaldehyde, and kept at 4 °C up to a month before staining. Brain sections were washed using 1X PBS before being blocked with vehicle (0.3% TritonX-100, 10% normal goat serum, 0.5% bovine serum albumin) for 1.5 h at room temperature, and incubated with conjugated antibody (200–542-211, Jackson ImmunoResearch, PA, USA) overnight at 4 °C. Slides were then washed with vehicle, 1X PBS, and 0.5X PBS before being placed and air dried on a slice, and cover-slipped with mounting media (Sigma). Images were taken with a confocal microscope (Leica LAS).

Thymidine (T1895), paraformaldehyde, sucrose, phosphate buffered saline (PBS), Tween 20, BSA, 4′,6‐diamidino‐2‐phenylindole (DAPI), mounting media, phenylmethanesulfonyl fluoride, deoxyribonuclease I from bovine pancreas, Hepes, MgCl₂, NaCl, glycerol, Triton X‐100, imidazole, 2‐mercaptoethanol, dUMP, NADPH, l‐serine and Duolink PLA kit (DUO92007) were purchased from Sigma‐Aldrich. THF was provided from Merck & Cie (Schaffhausen, Switzerland). Protein G‐Plus agarose beads (Sc‐2002) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Dulbecco’s phosphate buffered saline (20‐031‐CV), fetal bovine serum (35‐015‐CV) and 0.25% trypsin (25‐053‐CI) were purchased from Corning Inc. (Corning, NY, USA); BCA kit (quantumMicro Protein, EMP015480) was obtained from EuroClone (Pero, Italy).

FFPE tissue sections were deparaffinized in xylene for 10 minutes and rehydrated in a series of alcohol solutions (100% ethanol twice, 95% ethanol, 75% ethanol). The sections were stained with hematoxylin (Sigma), a nucleus dye, rinsed with tap water, and then counterstained in eosin (Sigma) which stains the cytoplasmic material. The sections were then dehydrated with alcohol solutions, cleared in xylene and then coverslipped using a mounting media (Sigma). With H&E staining, nuclei are stained blue or purple, whereas the cytoplasm is stained pink.

Reagents were obtained from the following sources: unlabeled amino acids (Sigma-Aldrich; St. Louis, MO, USA); TRIzol and Lipofectamine 2000 (Invitrogen-Gibco; Carlsbad, CA, USA); GeneAmp RT-PCR kit (Applied Biosystems/Life Technologies; Carlsbad, CA, USA); Taq polymerase kit (TaKaRa; Mountain View, CA, USA); Power Block (Biogenex; San Ramon, CA, USA); GSH-Glo Glutathione Assay Kit, pUIIR3-EGFP and pGL3-Basic vectors (Promega; Madison, WI, USA); rabbit polyclonal anti-human Nrf2 antibody (AbCam; Cambridge, MA, USA); Fluoromount mounting media (Sigma, St. Louis, MO, USA); Enhanced ChemiLuminescence kit (PerkinElmer Life Sciences, Waltham, MA, USA); Bio-Rad RC DC protein assay kit (Bio-Rad; Hercules, CA, USA); Pierce Pinpoint Cell Surface Isolation kit (Thermoscientific; Rockford, IL, USA); and [3H]-glutamate and [3H]-taurine (Perkin Elmer Life Sciences; Boston, MA, USA). Rabbit polyclonal anti-xCT antibody was a generous gift from Dr. Sylvia B. Smith [17 (link)]. Culture media and supplements, goat anti-rabbit Alexa Fluor 568 secondary antibody, and Hoechst 33342 stain were from Invitrogen-Gibco (Carlsbad, CA, USA).