. no. 610296, BD Biosciences, San Jose, CA) Mouse anti-NMDAR1 (1:25, cat. no. MAB

. no. 610296, BD Biosciences, San Jose, CA) Mouse anti-NMDAR1 (1:25, cat. no. MAB363, A-836339 site Chemicon, USA) (Lin Talman, 2002), mouse anti-GluR2 (1:25, cat. no. MAB397, Chemicon) (Lin et al. 2008), rabbit anti-PGP9.5 (1:100, cat. no. AB1961ASR, Chemicon) (Lin et al. 2010), rabbit anti-TH (1:100, cat. no. AB152, Chemicon), rabbit anti-VGluT1 and rabbit anti-VGluT2 (both from Dr R. H. Edwards, University of California San Francisco) (Fremeau et al. 2001; Lin Talman, 2006), mouse anti-GFAP (1:100, cat. no. G3893, Sigma-Aldrich, USA), mouse anti-NF160 (1:25, cat. no. MAB5254, Chemicon), mouse anti-macrophage (1: 100, cat. no. CBL260, Millipore, Billerica, MA, USA). Appropriate fluorescent secondary antibodies made in donkey against respective primary antibodies were also used in place of RRX-conjugated anti-sheep antibody. Multiple-label immunofluorescent staining was performed for those primary antibodies that were raised in different species in some sections to reduce the number of animals needed. In this case, primary antibodies were mixed in incubation medium as described in our earlier publications (Lin Talman, 2005; Lin et al. 2007; Lin et al. 2008). In addition, we performed nuclear staining of the NTS by incubating sections with 0.5 M TO-PRO-3 (Invitrogen) in PBS for 15 min. We analysed stained sections with a Zeiss LSM 510 or LSM 701 confocal laser-scanning microscope as described in our earlier publication (Lin et al. 2000; Lin Talman, 2002; Lin et al. 2004). Digital confocal images were obtained and processed with software provided with the Zeiss LSM 510 or LSM 710. We also performed Nissl stain and examined the stained sections with a Nikon Optiphot microscope.Image analysis for nNOS immunostainingWe quantified nNOS-IR in the NTS, NA, CVLM and RVLM with the NIH ImageJ software (a public domain program available from the NIH, http://rsb.info.nih.gov/ij/). These analyses included all nNOS-IR observed in cells and processes in each of the areas and normalized by the area selected for analysis. We also counted cells that were positive for nNOS-IR in the NTS, nucleus ambiguus (NA), nodose ganglionC2012 The Authors. The Journal of PhysiologyC2012 The Physiological SocietyJ Physiol 590.nNOS and the baroreflex(NG), caudal ventrolateral medulla (CVLM) and rostral ventrolateral medulla (RVLM). We used two to three sections from each animal for each of the areas analysed. NTS sections within 200 m of the centre of the injection site were used (Bregma -13.40 to -13.80 mm) for analysis. Sections selected for the NA were from Bregma -12.50 to -12.90 mm, RVLM from Bregma -12.70 to -13.10 mm, CVLM from Bregma -14.00 to -14.30 mm. Student’s two tailed t test was used to determine if nNOS-IR was statistically significantly different between AAV2nNOSshNOS and PBS control groups in different areas. Significance was accepted at P values 0.05.Delivery of vectors into NTS for baroreflex analysisWe bilaterally microGW0742 web injected (200 nl) AAV2nNOSshRNA into NTS or, for controls, bilaterally injected either phosphate buffered saline, AAV2nNOScDNA, or AAV2eGFP. Animals were all allowed to recover from surgery and to remain in their home cage for 2 weeks before returning to the lab for instrumentation and study of baroreflex responses. After injection and removal of the pipette, buprenorphine (0.01?.05 mg kg-1 ) was administered subcutaneously, wounds were closed and anaesthesia was stopped. After full recovery from anaesthesia each animal was returned to its.. no. 610296, BD Biosciences, San Jose, CA) Mouse anti-NMDAR1 (1:25, cat. no. MAB363, Chemicon, USA) (Lin Talman, 2002), mouse anti-GluR2 (1:25, cat. no. MAB397, Chemicon) (Lin et al. 2008), rabbit anti-PGP9.5 (1:100, cat. no. AB1961ASR, Chemicon) (Lin et al. 2010), rabbit anti-TH (1:100, cat. no. AB152, Chemicon), rabbit anti-VGluT1 and rabbit anti-VGluT2 (both from Dr R. H. Edwards, University of California San Francisco) (Fremeau et al. 2001; Lin Talman, 2006), mouse anti-GFAP (1:100, cat. no. G3893, Sigma-Aldrich, USA), mouse anti-NF160 (1:25, cat. no. MAB5254, Chemicon), mouse anti-macrophage (1: 100, cat. no. CBL260, Millipore, Billerica, MA, USA). Appropriate fluorescent secondary antibodies made in donkey against respective primary antibodies were also used in place of RRX-conjugated anti-sheep antibody. Multiple-label immunofluorescent staining was performed for those primary antibodies that were raised in different species in some sections to reduce the number of animals needed. In this case, primary antibodies were mixed in incubation medium as described in our earlier publications (Lin Talman, 2005; Lin et al. 2007; Lin et al. 2008). In addition, we performed nuclear staining of the NTS by incubating sections with 0.5 M TO-PRO-3 (Invitrogen) in PBS for 15 min. We analysed stained sections with a Zeiss LSM 510 or LSM 701 confocal laser-scanning microscope as described in our earlier publication (Lin et al. 2000; Lin Talman, 2002; Lin et al. 2004). Digital confocal images were obtained and processed with software provided with the Zeiss LSM 510 or LSM 710. We also performed Nissl stain and examined the stained sections with a Nikon Optiphot microscope.Image analysis for nNOS immunostainingWe quantified nNOS-IR in the NTS, NA, CVLM and RVLM with the NIH ImageJ software (a public domain program available from the NIH, http://rsb.info.nih.gov/ij/). These analyses included all nNOS-IR observed in cells and processes in each of the areas and normalized by the area selected for analysis. We also counted cells that were positive for nNOS-IR in the NTS, nucleus ambiguus (NA), nodose ganglionC2012 The Authors. The Journal of PhysiologyC2012 The Physiological SocietyJ Physiol 590.nNOS and the baroreflex(NG), caudal ventrolateral medulla (CVLM) and rostral ventrolateral medulla (RVLM). We used two to three sections from each animal for each of the areas analysed. NTS sections within 200 m of the centre of the injection site were used (Bregma -13.40 to -13.80 mm) for analysis. Sections selected for the NA were from Bregma -12.50 to -12.90 mm, RVLM from Bregma -12.70 to -13.10 mm, CVLM from Bregma -14.00 to -14.30 mm. Student’s two tailed t test was used to determine if nNOS-IR was statistically significantly different between AAV2nNOSshNOS and PBS control groups in different areas. Significance was accepted at P values 0.05.Delivery of vectors into NTS for baroreflex analysisWe bilaterally microinjected (200 nl) AAV2nNOSshRNA into NTS or, for controls, bilaterally injected either phosphate buffered saline, AAV2nNOScDNA, or AAV2eGFP. Animals were all allowed to recover from surgery and to remain in their home cage for 2 weeks before returning to the lab for instrumentation and study of baroreflex responses. After injection and removal of the pipette, buprenorphine (0.01?.05 mg kg-1 ) was administered subcutaneously, wounds were closed and anaesthesia was stopped. After full recovery from anaesthesia each animal was returned to its.

Leave a Reply