For immunohistochemistry, sections were stained for overnight at 4°C with prestin polyclonal rabbit antibody (1∶70 diluted, Santa Cruz, sc30163, USA) using the microwave antigen retrieval technique.
Protein concentration was determined with a Bradford assay kit (BioRad, Hercules, CA, USA) using albumin diluted in lysis buffer as the internal standard.
Analyses of ANAs were performed by indirect immunofluorescence on human epidermal cell 2 (HEp-2 cells) slides (Immunoconcept, Sacramento, CA, USA) using 1 100 diluted samples.
Briefly, RIA buffer [80% phosphate-buffered saline (PBS), 20% DMEM, 2% normal rabbit serum], 100 μL cold standard (rat PRL-RP-3) or unknown sample, rPRL-s-9 antiserum (final dilution of 1 437,500 in RIA buffer), and [I]-rat-PRL (PerkinElmer, Wellesley, MA, USA; using 15,000 counts per tube diluted in RIA buffer) were combined and incubated with shaking, overnight at 4°C.
Serum diluted to 50 % with or without 500,000 cpm = counts per minute I DNase-I (Bioworld, Dublin, OH, USA), labeled using the chloramine T method, was diluted in DNase buffer and separated on a Superose 12 column (GE healthcare) using the ÄKTA system (GE healthcare).
Whole mount immunocytochemistry was performed as previously described [37] except that PBS containing 5% skim milk (Nestlé Foods Inc., North York, ON, Canada) plus 0.1% Triton-X 100 (Sigma, St . Louis MO, USA) was used to dilute the primary antibody (rabbit anti-neurogranin at 1 1000).
Membranes were probed with appropriate primary antibodies followed by horseradish peroxidase (HRP -conjugated secondary antibodies (Cell Signaling, Danvers, MA, USA) diluted 1 : 5000, and visualized using tHRP -conjugatedmiluminesecondarytection system (ECL) antibodieslms (Thermo SCell Signalingksbury, MA, USA).
The gas chromatograph was calibrated using the dynamic gas dilution principle with several concentrations of gaseous isoprene using liquid isoprene (Sigma, USA) diluted in methanol and evaporated with a gas dilutor (MK5, MCZ Umwelttechnik, Germany).
The ammonium concentrations were measured in 15-fold diluted samples using the LCK 303 kit (Hach Lange, USA).
The labeling was visualized with horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG, diluted 1 5000, or anti-goat IgG, diluted 1 10,000; Sigma) using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
