7; Price 1985). Hence, within the present study, we aimed to assess and characterise CB1 receptor expression within the perikarya and peripheral and central processes of key nociceptive sensory neurons, which had been identified by markers in the two principal sub-populations of nociceptive principal sensory neurons, CGRP immunopositivity and IB4 binding (Price tag 1985; Silverman and Kruger 1988).NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptMaterials and methodsAnimals and tissue preparations All experiments have been carried out in accordance using the UK Animals (Scientific Procedures) Act 1986, the revised National Institutes of Health Guide for the Care and Use of Laboratory Animals, the European Communities Council Directive (86/609/EEC) plus the recommendations of the Com-mittee for Research and Ethical Challenges of IASP, published in Discomfort, 16 (1983) 109?ten. Moreover, all experiments have been authorized by the Ethics Committee from the University of Debrecen, Debrecen, Hungary, as well as the Animal Subjects Review Board in the University of Porto, Porto, Portugal. All efforts have been made to minimise the amount of animals employed inside the present study. Altogether, ten rats had been killed (Wistar-Kyoto, 250?00 g; six rats were utilised for immunohistochemistry and 2 and two animals for DNA and protein preparation for RT-PCR and Western blot, respectively). Also, 3 wild-type (C57BL/6 J) and three CB1-/- mice (gift in the Institute of Experimental Medicine on the Hungarian Academy of Sciences, Budapest, Hungary, transfer approved by Dr. Andreas Zimmer) were employed within the experiments. For reverse transcriptase polymerase chain reaction (RT-PCR), samples of the hippocampus, L4? dorsal root ganglia (DRG), dorsal and ventral 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 were immersed in RNA-later (Ambion, Inc., TX, USA) quickly right 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 were removed in the killed rats. The samples were pulverised in liquid nitrogen and solubilised in ice-cold 20 mM TRIS, 1 mM EDTA (pH 7.five) containing 1 NP-40, 0.5 deoxycholic acid and 0.2-Bromooxazole Data Sheet 1 SDS, supplemented with protease inhibitors (0.2-Bromo-3-fluoropyrazine Purity 1 mg/ml benzami-dine, 1 mM phenyl-methyl-sulfonyl fluoride, 5 /ml leupeptin, five /ml pepstatin A and 5 /ml aprotinin).PMID:32695810 Just after two h of gentle rocking at 4 , cellular debris have been removed by centrifugation (16,000 rpm, 10 min). The supernatant was stored at -20 till Western blotting. For immunohistochemistry, six rats and 3 wild-type and three CB1 receptor-/- mice had been deeply anesthetized with pentobarbital (100 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 four? DRG, the L4? spinal cord segment, the hippocampus, the urinary bladder and glabrous hindpaw skin had been removed and kept within the identical fixative for four h at 4 . Tissue blocks were 1st immersed in 20 after which 40 sucrose dissolved in 0.1 M PB till they sank. Fifty sections from DRG, spinal cord and hippocampus and 40-Brain Struct Funct. Author manuscript; obtainable in PMC 2014 May well 01.Veress et al.Pagesections in the urinary bladder have been reduce on a cryostat and stored in 30 sucrose at four until immunostaining.NIH-PA Author Manuscript NIH-PA.