Pectively). In addition, 3 wild-type (C57BL/6 J) and 3 CB1-/- mice (gift from the Institute of Experimental Medicine with the Hungarian Academy of Sciences, Budapest, Hungary, transfer authorized by Dr. Andreas Zimmer) had been employed within the experiments. For reverse transcriptase polymerase chain reaction (RT-PCR), samples on the hippocampus, L4 dorsal root ganglia (DRG), dorsal and ventral a part of the L4 spinal cord, glabrous hindpaw skin and urinary bladder were collected from killed rats (intraperitoneal pentobarbital injection; 100 mg/kg). Tissue samples have been immersed in RNA-later (Ambion, Inc., TX, USA) promptly after the dissection and stored at -20 until use. For Western blotting, L4 spinal cord, L4 DRG, glabrous hindpaw skin, urinary bladder and hippocampus samples had been removed from the killed rats. The samples had been pulverised in liquid nitrogen and solubilised in ice-cold 20 mM TRIS, 1 mM EDTA (pH 7.5) containing 1 NP-40, 0.5 deoxycholic acid and 0.1 SDS, supplemented with protease inhibitors (0.1 mg/ml benzami-dine, 1 mM phenyl-methyl-sulfonyl fluoride, five /ml leupeptin, 5 /ml pepstatin A and 5 /ml aprotinin). After 2 h of gentle rocking at four , cellular debris were removed by centrifugation (16,000 rpm, ten min). The supernatant was stored at -20 till Western blotting. For immunohistochemistry, six rats and three wild-type and 3 CB1 receptor-/- mice have been deeply anesthetized with pentobarbital (one hundred mg/kg) and transcardially perfused with Tyrode’s remedy followed by four paraformaldehyde, in 0.1 M phosphate buffer (0.1 M PB, pH = 7.four). The lumbar 4 DRG, the L4 spinal cord segment, the hippocampus, the urinary bladder and glabrous hindpaw skin have been removed and kept in the same fixative for four h at 4 . Tissue blocks were very first immersed in 20 and after that 40 sucrose dissolved in 0.1 M PB till they sank. Fifty sections from DRG, spinal cord and hippocampus and 40-Brain Struct Funct.Phlorizin Author manuscript; available in PMC 2014 Could 01.Trimethoprim Veress et al.Pagesections in the urinary bladder have been reduce on a cryostat and stored in 30 sucrose at four till immunostaining.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptRT-PCR Total RNA was isolated applying the RNeasy micro kit (Qiagen GmbH, Germany) as outlined by the manufacturer’s instructions. For first-strand cDNA synthesis, the Omni-script RT kit (Qiagen GmbH, Germany) was applied with 1 of total RNA and oligo d(T) primer. CB1specific primers have been developed employing the rat CB1 gene (accession no. NM_102784 in the NCBI database). To distinguish amplification of genomic DNA, the primer pairs were placed in the 5 UTR of exon 1 and exon two, respectively. The primer sequences were: five CAAGCAAGGAGCACC CAT (sense primer) and five TGAAGGAGGCTGTAACCC (antisense primer).PMID:24732841 The predicted solution size was 691 bp. Amplification of -actin was applied as a handle for the reaction. The amplicon for -actin had a 317 bp predicted length, and also the primer sequences have been as follows: actin sense 5-TGCGTGACATTAAAGAGAAG-3 and actin antisense 5-CTGCATCCTGTCAGCAATG-3. The reaction was performed with Tth polymerase (Promega Corp., WI, USA) and included a 4-min incubation at 95 , followed by 30 cycles of 40 s at 94 , 1 min at 58 , 1.5 min at 70 in addition to a final extension step of 5 min at 70 . The amplified PCR fragments have been separated on 1.five agarose gel (SigmaAldrich Co., MO, USA) in 1 BE buffer and stained with acridine-orange dye and documented by a MiniBis Pro geldoc Technique (DNR Bio-Im.
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