The blots indicated that Novus 18801-50 was far more specific than Santa Cruz SC-1173; hence it was the only antibody used for immunohistochemistry. immunolesioned rats were treated with the selective AT1 antagonist, Candesartan. Results Immunohistochemistry and autoradiography revealed AT1 expression in dorsal BMS-983970 root ganglia, superior cervical ganglion. In the dorsal horn of the spinal cord, AT1 immunostainining and angiotensin binding were both prominent. In ventral horn and IML, immunoreactivity for AT1 and choline acetyltransferase co-localized in pre-ganglionic sympathetic and somatic motor neurons. Immunolesion caused CXCL5 over 50% loss of IML perikarya within 3 months. Concurrent treatment with BMS-983970 the AT1 antagonist, Candesartan, did not affect the outcome. Discussion AT1 expression is surprisingly widespread in sensory, autonomic and somatic motor neurons of the rat. This expression may be important to the normal physiology of these systems. Present data, however, do not support the concept that AT1 activation contributes to the loss of autonomic neurons after axonal damage. under protocols approved by the Mayo Institutional Animal Care and Use Committee. For immunolesion, we used murine monoclonal ZR 2, 3, 4 and 6 antibodies12, which target multiple epitopes of rat acetylcholinesterase (AChE). An equal-parts antibody mixture (1.6 mg protein in 2 ml 0.9% NaCl) was injected into the tail vein without anesthesia. Drug treatment Candesartan stock solution contained 100 mg drug mixed with vehicle: 10 ml polyethylene glycol 400 (Sigma-Aldrich), 5 ml ethanol, 2 ml cremophor-EL (Sigma), and water to a final volume of 100 ml. Every 2C3 days, a working quantity of stock (pH9) was freshly diluted into drinking water by a factor of 10 or more, calculated to deliver average daily doses of 10 mg/kg based on measured water intake per cage. Rats started on Candesartan 1 week before antibody treatment and continued with access for 40 days. There were six experimental groups in the immunolesion study: (1) normal drinking water and no further treatment ( em n /em =8); (2) drug vehicle and no further treatment ( em n /em =6); (3) Candesartan but no other treatment ( em n /em =5); (4) AChE antibody ( em n /em =11); (5) drug vehicle followed by antibody ( em n /em =6); (6) Candesartan followed by antibody ( em n /em =6). Western blotting In preparation for immunohistochemistry, we tested the specificity of AT1 antibodies by Western blot analysis on rat spinal cord tissues. Approximately 0.1 g samples of thoracic spinal cord were dissected and homogenized in 3 ml homogenization buffer: 10 mM EDTA, 300 mM sucrose, 1% SDS, 0.1 mM PMSF and 450 l protease BMS-983970 inhibitor cocktail stock (Roche, 1836170). Samples were centrifuged at 12,000 rev/min for 10 minutes, supernatants were heated at 100C for 5 minutes, and 20 l aliquots were separated on 10% Tris-HCl SDS-Ready gels (BioRad, 161-1155) at 100 V for 1 hour. Proteins were then transferred onto Immobilon-P Transfer Membrane (Millipore IPVH00010 from Fisher) in transfer buffer (Bio-Rad 161-0734) at 200 mA for 1.5 hour at 4C. Following transfer, the membranes were blocked BMS-983970 with 5% non-fat milk for 1 hour at room temperature, followed by incubation with AT1 antibodies from Novus (Littleton Co., Ab 18801-50) and Santa Cruz (SC-1173) at 1 : 500 dilutions overnight at 4C. After washing thoroughly with 0.1% Tween in PBS, membranes were incubated with AP conjugated goat-anti-rabbit IgG (Santa Cruz, SC-2007) at 1 : 3000 for 1.5 hours at room temperature. After another several washes with 0.1% Tween in PBS, immunoreactivity was visualized by development in Nitroblue Tetrazolium Chloride (Roche, 1087-479) and 5-Bromo-4-chloro-3-indolyl BMS-983970 phosphate (Roche, 760-994). Immunohistochemistry Rats under pentobarbital anesthesia (45 mg/kg) were perfused through the heart with phosphate-buffered saline (PBS), 0.1M, pH 7.4, followed by 4% paraformaldehyde in PBS. Representative segments of thoracic spinal cord (T1, T2 and T8) were located by dorsal root entries. Tissues were post-fixed.