t. injec tions, Ang II and losartan have been dissolved in Ringers solu tion. PD123319, U0126, SB203580 and SP600125 have been dissolved in Ringers option containing 6. 8% dimethyl sulfoxide, Once the effects of Ang II receptor antagonists and MAPK linked inhibitors were tested, they were co injected with Ang II within a volume of five ul. Mor phine was dissolved in physiological saline and adminis tered intraperitoneally five min prior to injection of Ang II. Immunohistochemical staining Spinal cords for measurement of AT1 receptors had been pre pared inside 24 h following delivery. Mice have been anesthe tized with sodium pentobarbital and perfused with the heart with ice cold phosphate buffered saline, instantly followed by a fixative containing 4% paraformaldehyde and 0. 2% glutaraldehyde in PBS.
Spinal cords had been then postfixed together with the exact same fixative resolution at 4 C for 1 h then placed in a 20% sucrose buffered solution at 4 C for twelve h. Tissues have been frozen on dry ice and minimize into 20 um thick coronal sections on the cryostat, The immunohistochemical selleck chemical staining method was carried out as previously described, Briefly, a rabbit anti AT1 receptor antibody, Millipore Co, USA was utilized to spinal cord slices, which were then incu bated at 4 C for twelve h. The secondary antibody consisted of FITC labeled anti rabbit IgG goat serum, and was allowed to react from the dark at space temperature for 2 h. The stained sections have been mounted in Dako Fluorescence Mounting Medium, and stored at 4 C in the dark room till measurements had been carried out.
The distribu tion of AT1 receptor immunofluorescence intensities was quantitatively analyzed applying a MapAnalyzer, The background value, together with non distinct fluorescence originating from glutaraldehyde, was subtracted photometrically from the complete fluores cence intensity worth at each level selleck chemicals MK-0752 measured. SDS polyacrylamide gel electrophoresis and immunoblotting Samples used for immunoblotting have been ready as fol lows. At 10 min just after i. t. injection, mice were decapi tated as well as entire spinal cord was taken by pressure expulsion with physiological saline. The dorsal a part of lumbar spinal cord was dissected quickly on ice cooled glass dish. The tissue samples had been homoginaized in 0. 15 ml of CelLytic MT Manmalian Tissue Lysis Extrac tion Reagent and centrifuged the lysis sample at 15,000? g for 15 min at 4 C. Supernatants had been dissolved in four ? Laemmli sample buffer, and boiled at 95 C for ten min. Electrophoresis was performed on 10% acrylamide gels. Proteins had been transferred electrically in the gel onto a polyvinylidene difluoride membrane from the semi dry blotting method. The blots have been blocked for 30 min with 5% skim milk in Tris buffered sa line supplemented with 0.