Glutamine synthetase

Transduced mouse retinas were dissected and fixed in 4% paraformaldehyde for 30 min at room temperature. The retinas were incubated in PBS with 1% Triton X-100, 0.5% Tween 20 for 1 h at room temperature and in 10% normal goat serum for 1 h at room temperature and then incubated overnight at 4 °C with primary antibody: Brn3a (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rhodopsin (1:500; Abcam, Cambridge, UK), PKCα (1:500; Abcam), calbindin (1:500; Abcam) and glutamine synthetase (GS) (1:500; Abcam) in blocking buffer. Secondary anti-rabbit IgG and anti-mouse IgG conjugated with Alexa TM594 and Alexa TM555, respectively (1:1,000; Molecular Probes), were applied for 1 h at room temperature. The retinas then were flat-mounted and the sections mounted on slide glass.

Fluorescent immunohistochemistry was performed on cryostat sections using the method as described previously [23 (link)]. Primary mouse monoclonal, rat monoclonal or rabbit polyclonal antibodies raised against calbindin (Swant, Marly, Switzerland; 1:1000), CD68 (Serotec, Raleigh, NC; 1:500), glial fibrillary acidic protein (GFAP; DAKO, Carpinteria, CA; 1:300), glutamine synthetase (Abcam, Cambridge, MA; 1:1000), alpha subunit of G protein (Goα; Millipore, Billerica, MA; 1:1000), hexaribonucleotide binding protein-3 (NeuN; Millipore; 1:100), Neurofilament (SMI32; Covance, Munich, Germany; 1:1000), Npc1 (Abcam; 1:300), alpha isoform of protein kinase C (PKCα; Sigma; 1:1000), tyrosine hydroxylase (Millipore; 1:1000), and a microglia marker (F4/80; 1:200) were used. All primary antibodies are specific to determine their target proteins, which were used as specific markers to identify different types or structures of cells. Alexa 488-labeled (Molecular Probes, Eugene, USA) or Cy3-labeled (Dianova, Hamburg, Germany) secondary antibodies against mouse, rat or rabbit IgG were used. Fluorescence in sections was detected by a confocal microscope (Zeiss LSM780; Jena, Germany). Digital images were adjusted in contrast and brightness with the Photoshop software (Adobe Systems, San Jose, USA).

Snap-frozen heart samples were homogenized and lysed in a buffer containing 25 mM Tris·HCl, 150 mM NaCl, 2 mM EGTA, 5 mM EDTA, 0.5% NP-40, and a protease and phosphatase inhibitor cocktail (Sigma-Aldrich). Protein concentration was determined using the Bradford reagent (Sigma-Aldrich). Tissue homogenates were separated by SDS-PAGE and transferred to nitrocellulose membranes followed by Western blot analysis. Antibodies used were as follows: glucose transporters 1 and 4 (GLUT1 and GLUT4; Abcam), LDHA (Proteintech), hexokinase 1 (Abcam), PDK4 (Abcam), pyruvate dehydrogenase E1α (PDH-E1α; Abcam), phospho-PDH-E1α (S293, p-PDH-E1α; Abcam), pyruvate carboxylase (Santa Cruz), glutamine synthetase (Abcam), glutaminase (Abcam), O-linked β-N-acetylglucosamine (GlcNac RL2; Abcam), glutamine fructose amidotransferase-2 (GFAT2; Abcam), and GAPDH (Sigma-Aldrich).