Western blotting experiments
Nervous tissues [brain, spinal cord, and dorsal root ganglia (DRG)], isolated from adult male Sprague–Dawley rats were homogenized separately with a Polytron in 50 mM Tris–HCl, pH 7.5, and 5 mM EDTA containing 1% Triton X-100 and protease inhibitors (Bioshop, Burlington, ON, Canada), and centrifuged at 24 000 g for 30 min at 4oC. The proteins from the supernatant were subsequently denatured using Laemmli sample buffer (0.375 mM Trisma base, pH 6.8, 12% w/v sodium dodecyl sulfate, 30% v/v glycerol, 20% v/v b-mercaptoethanol, and 0.2% w/v bromophenol blue) and equal amounts of proteins (25 lg) were resolved using 10% sodium dodecyl sulfate–polyacrylamide gels. Proteins were then electroblotted onto polyvinylidene difluoride membranes for 1 h at 22C (Roche Molecular Biochemicals, Laval, QC, Canada). A pre-stained Dual color molecular mass marker was used to standardize gel migration (Biorad, Mississauga, ON, Canada). Polyvinylidene difluoride membranes were incubated with 0.05% Tween 20 and 6% powdered skim milk in Tris-buffered saline (TBS; pH 7.5) 2 h at 22oC to block non-specific sites. Membranes were immunoblotted overnight at 4C with the specific anti-NTS1 receptor guinea-pig antibody (1 : 5000; Neuromics Edina, MN, USA) in blocking solution. After washing with TBS–Tween 20 (0.05%), blots were incubated for 1 h at 22oC with horseradish peroxidase-conjugated secondary anti-guinea pig antibody (1 : 5000; Sigma-Aldrich, St Louis, MO, USA) in TBS– Tween 0.05% with 3% powdered skim milk. Proteins were then visualized using an enhanced chemiluminescent detection kit (PerkinElmer, Boston, MA, USA). Blots were digitized by scanning with an HP Scanjet 4070 Photosmart (Mississauga, ON, Canada)\and image processing was performed using Photoshop version 8.0.1 imaging software (Adobe Systems, San Jose, CA, USA) on an MAC G5-compatible computer (INSO, Sherbrooke, QC, Canada).
Light microscopic immunodetection of NTS1 receptors in vivo
Adult Sprague–Dawley rats were deeply anesthetized with ketamine/ xylazine (87/13 mg/kg) administered intramuscularly (300 lL) and perfused transaortically with a freshly prepared solution of 4% p-formaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. Tissues were rapidly removed, cryoprotected overnight in 0.1 M PB, containing 30% sucrose at 4oC, and frozen for 1 min in isopentane at )40oC. Spinal cords were sectioned transversely at 30 lm on a Leica SM2000R sliding microtome (Leica, Dollard-des-Ormeaux, QC, Canada) and DRG were cut on a Leica CM1850 cryostat at 20 lm. Immunostaining was performed according to the avidin–biotin peroxidase method (Elite ABC kit; Vector Laboratories, Burlingame, ON, Canada) using a nickel-intensified diaminobenzidine protocol to localize the horseradish peroxidase immunoreaction product as described previously (Sarret et al. 2003). Briefly, tissue sections were washed twice with 0.1 MTBS, pH 7.4, pre-treated for 30 min with3% hydrogen peroxide in 0.1 M TBS to quench endogenous peroxidase. Serial sections were then washed twice with TBS (2 · 10 min), preincubated for 1 h at 22C in a blocking solution containing 3% normal goat serum (NGS) and 0.2% Triton X-100 in TBS, and incubated overnight at 4oC with the primary NTS1 antibody (1/1000) diluted in TBS containing 0.05% Triton X-100 and 0.5% NGS. Following two rinses in TBS containing 1% NGS (2 · 10 min), sections were incubated for 1 h at 220C in biotinylated goat anti-guinea pig immunoglobulin diluted 1/400 in TBS (Vector laboratories, Burlington, ON, Canada), and then 1 h in Elite ABC solution (Vector Laboratories; prepared according to the manufacturer’s instructions). Visualization of bound peroxidase was achieved by reaction in a solution of 0.1 M Tris–HCl (TB; pH 7.4) containing 0.05% 3-3'-diaminobenzidine (DAB; Sigma-Aldrich), 0.04% nickel chloride, and 0.001% H2O2. The same procedure was used for control experiments except that sections were processed either with antibodies pre-adsorbed overnight in an excess of immunizing peptide or in the absence of primary antibody. The NTS1 immunoreactivity was completely abolished by pre-adsorption of the antibody with 2 lg/mL of blocking antigen. Reacted sections were mounted on Super frost plus slides (VWR, Mississauga, ON, Canada), dehydrated in graded ethanol, defatted in xylene, and mounted with Permount (Fisher Scientific, Montreal, QC, Canada). Labeled structures were examined under bright-field illumination with a Leica DM-4000 automated research microscope (Leica) and digitized images were obtained with a Lumenera InfinityX-21 digital camera using Infinity Capture software (Lumenera Corporation, Ottawa, ON, Canada).
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