rabbit polyclonal anti-nos1 (r20)  (Santa Cruz Biotechnology)

Journal: PLoS ONE

Article Title: Carbon Monoxide Reduces Neuropathic Pain and Spinal Microglial Activation by Inhibiting Nitric Oxide Synthesis in Mice

doi: 10.1371/journal.pone.0043693

Figure Lengend Snippet: The protein expression in the ipsilateral site of the lumbar section of the spinal cord of NOS1 (A) and NOS2 (B) from sciatic nerve-injured (CCI) WT and NOS2-KO mice treated with vehicle, CORM-2 or CoPP at 20 days after surgery is represented. The expression of NOS1 and NOS2 in the spinal cord from sham-operated WT and NOS2-KO mice treated with vehicle has been also represented as controls (sham-vehicle). In both figures and genotypes, *indicates significant differences when compared vs. their respective sham-operated vehicle treated mice (*p<0.05, one-way ANOVA followed by the Student Newman Keuls test). Representative examples of western blot for NOS1 (155 kDa) and NOS2 (130 kDa) proteins in which β-actin (45 kDa) was used as a loading control are also shown. Data are expressed as mean values ± SEM; n = 5 samples per group.

Article Snippet: The proteins were electrophoretically transferred onto PVDF membrane for 120 minutes for HO-1, HO-2 and CD11b/c or over night for NOS1 and NOS2 detection, blocked with PBST +5% nonfat dry milk, and subsequently incubated overnight at 4°C with a polyclonal rabbit anti-HO-1 (1∶300, Stressgen, Ann Arbor, MI), a polyclonal rabbit anti-HO-2 (1∶1000, Stressgen, Ann Arbor, MI), a polyclonal rabbit anti-CD11b/c (1∶300, Novus Biologicals) antibody against the type 3 complement receptor to detect activated microglial cells , a polyclonal rabbit anti-NOS1 antibody (1∶100, BD Transduction Laboratories, San Diego, CA, USA) or a polyclonal rabbit anti-NOS2 antibody (1∶200, Chemicon, Millipore).

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: Carbon Monoxide Reduces Neuropathic Pain and Spinal Microglial Activation by Inhibiting Nitric Oxide Synthesis in Mice

doi: 10.1371/journal.pone.0043693

Figure Lengend Snippet: A summary of the results from protein expression studies obtained in the ipsilateral site of the dorsal root ganglia (DRG) and/or the lumbar section of the spinal cord (SC) from sciatic nerve-injured (CCI) WT and NOS2-KO mice treated with vehicle, CORM-2 or CoPP is shown.

Article Snippet: The proteins were electrophoretically transferred onto PVDF membrane for 120 minutes for HO-1, HO-2 and CD11b/c or over night for NOS1 and NOS2 detection, blocked with PBST +5% nonfat dry milk, and subsequently incubated overnight at 4°C with a polyclonal rabbit anti-HO-1 (1∶300, Stressgen, Ann Arbor, MI), a polyclonal rabbit anti-HO-2 (1∶1000, Stressgen, Ann Arbor, MI), a polyclonal rabbit anti-CD11b/c (1∶300, Novus Biologicals) antibody against the type 3 complement receptor to detect activated microglial cells , a polyclonal rabbit anti-NOS1 antibody (1∶100, BD Transduction Laboratories, San Diego, CA, USA) or a polyclonal rabbit anti-NOS2 antibody (1∶200, Chemicon, Millipore).

Techniques: Expressing

Journal: PLoS ONE

Article Title: The Spinal Cord Expression of Neuronal and Inducible Nitric Oxide Synthases and Their Contribution in the Maintenance of Neuropathic Pain in Mice

doi: 10.1371/journal.pone.0014321

Figure Lengend Snippet: Development of mechanical allodynia ( A ), thermal hyperalgesia ( B ) and thermal allodynia ( C ) in the ipsilateral paw of WT, NOS1-KO and NOS2-KO mice at 0, 1, 4, 7, 10, 14 and 21 days after sciatic nerve ligation. For each test and time tested, * indicates significant differences when compared sciatic nerve-injured WT mice vs. sham-operated WT mice (* p<0.05, ** p<0.01, *** p<0.001, one-way ANOVA followed by Scheffe test), + when compared sciatic nerve-injured NOS1-KO mice vs. sciatic nerve-injured WT mice (+ p<0.05, ++ p<0.01, +++ p<0.001, one-way ANOVA followed by Scheffe test) and # when compared sciatic nerve-injured NOS2-KO mice vs. sciatic nerve-injured WT mice (# p<0.05, ## p<0.01, ### p<0.001, one-way ANOVA followed by Scheffe test). In the cold plate test (C), • indicates significant differences when compared sciatic nerve-injured NOS1-KO mice vs. sham-operated NOS1-KO mice (• p<0.05, •• p<0.01, one-way ANOVA followed by Scheffe test). Results are shown as mean values ± SEM; n = 10–12 animals per experimental group.

Article Snippet: The proteins were electrophoretically transferred onto PVDF membrane overnight, blocked with PBST +2% nonfat dry milk, and subsequently incubated overnight at 4°C with a polyclonal rabbit anti-NOS1 antibody (1∶150, BD Transduction Laboratories, San Diego, CA, USA), a polyclonal rabbit anti-NOS2 antibody (1∶200, Chemicon, Millipore), a polyclonal rabbit anti-NOS3 antibody (1∶100, BD Transduction Laboratories, San Diego, CA, USA), or a monoclonal rabbit anti-β-actin antibody (1∶10.000, Sigma, St. Louis, MO, USA). β-actin was used as a loading control.

Techniques: Ligation, Mouse Assay

Journal: PLoS ONE

Article Title: The Spinal Cord Expression of Neuronal and Inducible Nitric Oxide Synthases and Their Contribution in the Maintenance of Neuropathic Pain in Mice

doi: 10.1371/journal.pone.0014321

Figure Lengend Snippet: The relative NOS1 mRNA ( A ) and protein ( B ) expression in the ipsilateral site of the lumbar section of the spinal cord from sham-operated (SH) and sciatic nerve-injured (SNI) WT and NOS2-KO mice at 21 days after sciatic nerve ligation are represented. In both figures, * indicates significant differences when compared sciatic nerve-injured WT mice vs. sham-operated WT animals (* p<0.05, one-way ANOVA followed by Student-Newman-Keuls test). A representative example of Western blots for NOS1 protein (155 kDa) in which β-actin (43 kDa) was used as a loading control is shown in C . Data are expressed as mean values ± SEM; n = 4–5 samples per group.

Article Snippet: The proteins were electrophoretically transferred onto PVDF membrane overnight, blocked with PBST +2% nonfat dry milk, and subsequently incubated overnight at 4°C with a polyclonal rabbit anti-NOS1 antibody (1∶150, BD Transduction Laboratories, San Diego, CA, USA), a polyclonal rabbit anti-NOS2 antibody (1∶200, Chemicon, Millipore), a polyclonal rabbit anti-NOS3 antibody (1∶100, BD Transduction Laboratories, San Diego, CA, USA), or a monoclonal rabbit anti-β-actin antibody (1∶10.000, Sigma, St. Louis, MO, USA). β-actin was used as a loading control.

Techniques: Expressing, Ligation, Western Blot

Journal: PLoS ONE

Article Title: The Spinal Cord Expression of Neuronal and Inducible Nitric Oxide Synthases and Their Contribution in the Maintenance of Neuropathic Pain in Mice

doi: 10.1371/journal.pone.0014321

Figure Lengend Snippet: The relative NOS2 mRNA ( A ) and protein ( B ) expression in the ipsilateral site of the lumbar section of the spinal cord from sham-operated (SH) and sciatic nerve-injured (SNI) WT and NOS1-KO mice at 21 days after sciatic nerve ligation are represented. In both figures, * indicates significant differences when compared sciatic nerve-injured NOS1-KO mice vs. sham-operated NOS1-KO animals (* p<0.05, one-way ANOVA followed by Student-Newman-Keuls test). A representative example of Western blots for NOS2 protein (130 kDa) in which β-actin (43 kDa) was used as a loading control is shown in C . Data are expressed as mean values ± SEM; n = 4–5 samples per group.

Article Snippet: The proteins were electrophoretically transferred onto PVDF membrane overnight, blocked with PBST +2% nonfat dry milk, and subsequently incubated overnight at 4°C with a polyclonal rabbit anti-NOS1 antibody (1∶150, BD Transduction Laboratories, San Diego, CA, USA), a polyclonal rabbit anti-NOS2 antibody (1∶200, Chemicon, Millipore), a polyclonal rabbit anti-NOS3 antibody (1∶100, BD Transduction Laboratories, San Diego, CA, USA), or a monoclonal rabbit anti-β-actin antibody (1∶10.000, Sigma, St. Louis, MO, USA). β-actin was used as a loading control.

Techniques: Expressing, Ligation, Western Blot

Journal: PLoS ONE

Article Title: The Spinal Cord Expression of Neuronal and Inducible Nitric Oxide Synthases and Their Contribution in the Maintenance of Neuropathic Pain in Mice

doi: 10.1371/journal.pone.0014321

Figure Lengend Snippet: The relative NOS3 mRNA ( A ) and protein ( B ) expression in the ipsilateral site of the lumbar section of the spinal cord from sham-operated (SH) and sciatic nerve-injured (SNI) WT, NOS2-KO and NOS1-KO mice at 21 days after sciatic nerve ligation are represented. In both figures, * indicates significant differences when compared sham-operated NOS1-KO animals vs. the other groups. (* p<0.05, one-way ANOVA followed by Student-Newman-Keuls test). A representative example of Western blot for NOS3 protein (140 kDa) in which β-actin (43 kDa) was used as a loading control is shown in C . Data are expressed as mean values ± SEM; n = 4–5 samples per group.

Article Snippet: The proteins were electrophoretically transferred onto PVDF membrane overnight, blocked with PBST +2% nonfat dry milk, and subsequently incubated overnight at 4°C with a polyclonal rabbit anti-NOS1 antibody (1∶150, BD Transduction Laboratories, San Diego, CA, USA), a polyclonal rabbit anti-NOS2 antibody (1∶200, Chemicon, Millipore), a polyclonal rabbit anti-NOS3 antibody (1∶100, BD Transduction Laboratories, San Diego, CA, USA), or a monoclonal rabbit anti-β-actin antibody (1∶10.000, Sigma, St. Louis, MO, USA). β-actin was used as a loading control.

Techniques: Expressing, Ligation, Western Blot

Journal: bioRxiv

Article Title: Calcium transients in nNOS neurons underlie distinct phases of the neurovascular response to barrel cortex activation in awake mice

doi: 10.1101/2022.10.03.510654

Figure Lengend Snippet: A : GCaMP7f was expressed in NOS1 cre mice using systemic administration of an AAV-PHP.eB viral vector. The localization of GCaMP7f expression in nNOS neurons in the cortical layers was verified by immunohistochemistry (IHC). Right panels are enlargements of the dashed squares in the left panel (n=2,744 neurons in 5 mice). B : GCaMP7f expression in inhibitory (GAD67) or excitatory (CaMKII) neurons of nNOS cre mice treated with the AAV-PHP.eB viral vector. C : Neurons (%) expressing nNOS, GCaMP7f or both, and, among GCaMP7f positive neurons, those expressing GAD67 or CaMKII (n=690 neurons in 3 mice). D : Single-cell RNAseq data (mousebrain.org/development/downloads.html) showing expression of Nos1 both in excitatory ( Slc17a7 ) and inhibitory ( Gad1 ) neurons (%), confirming the finding by immunohistochemistry ( B ). Scale bar=50μm throughout.

Article Snippet: The following primary antibodies were used: rabbit anti-NOS1 polyclonal antibody (1:300, AB5380, Sigma-Aldrich), chicken anti-GFP polyclonal antibody (1:500, A10262, Invitrogen), mouse anti-GAD67 monoclonal antibody (1:300, MAB5406, Sigma-Aldrich), rabbit anti-CaMKII monoclonal antibody (1:300, ab52476, abcam).

Techniques: Plasmid Preparation, Expressing, Immunohistochemistry

Journal: Investigative Ophthalmology & Visual Science

Article Title: Gradual Increase in Environmental Light Intensity Induces Oxidative Stress and Inflammation and Accelerates Retinal Neurodegeneration

doi: 10.1167/iovs.61.10.1

Figure Lengend Snippet: Antibodies Used for Immunofluorescence and Western Blotting

Article Snippet: Nitric oxide synthase 1 (NOS1) antibody R-20 , Rabbit polyclonal , Santa Cruz Biotechnology , sc-648 , WB 1:200.

Techniques: Immunofluorescence, Western Blot, Marker, Binding Assay

Journal: Neuron

Article Title: Temporally and Spatially Distinct Thirst Satiation Signals

doi: 10.1016/j.neuron.2019.04.039

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal anti-GAD65+GAD67 Abcam Cat# {"type":"entrez-nucleotide","attrs":{"text":"Ab183999","term_id":"62086416","term_text":"AB183999"}} Ab183999 Rabbit polyclonal anti-Nos1 Santa Cruz Cat#sc-648; RRID: AB_630935 Chicken polyclonal anti-GFP Abcam Cat#ab13970; RRID: AB_300798 Bacterial and Virus Strains AAV2-Ef1a-DIO-eYFP UNC Vector Core N/A AAV2-Ef1a-DIO-ChR2-eYFP UNC Vector Core N/A AAV1-hSyn1-flex-GCaMP6s-WPRE-SV40 Penn Vector Core, Addgene Cat#100845-AAV1 AAV9-hSyn-dLight1.3 Dr. Lin Tian (UC, Davis) N/A AAV2-hSyn-DIO-hM3D(Gq)-mCherry UNC Vector Core N/A AAV1-hSyn1-SIO-stGtACR2-FusionRed (diluted 10 times in PBS before injection) Dr. David Anderson (Caltech) N/A Chemicals, Peptides, and Recombinant Proteins Silicone Oil Sigma-Aldrich 378348 Exendin-4 Sigma-Aldrich E7144 Clozapine N-oxide (CNO) Sigma-Aldrich C0832 D-Mannitol Sigma-Aldrich M9647 0.9% Sodium Chloride, USP Hospira NDC 0409–4888-02 Ensure Abbott N/A 20% Intra-lipid Sigma-Aldrich I141 Glucose Macron 4912–12 Blue dye Butler’s N/A Experimental Models: Organisms/Strains Mouse: wild-type (C57BL/6J) The Jackson Laboratory Strain#000664; RRID: IMSR_JAX:000664 Mouse: Nos1-Cre knockin (B6.129-Nos1 tm1(cre)Mgmj /J) The Jackson Laboratory Strain# 017526; RRID: IMSR_JAX:017526 Mouse: Ai75D (B6.Cg-Gt(ROSA) 26Sor tm75.1(CAG-tdTomato*)Hze /J) The Jackson Laboratory Strain# 025106; RRID: IMSR_JAX:025106 Mouse: Ai9 (B6.Cg-Gt(ROSA) 26Sor tm9(CAG-tdTomato)Hze /J) The Jackson Laboratory Strain# 007909; RRID: IMSR_JAX: 007909 Mouse: Ai3 (B6.Cg-Gt(ROSA) 26Sor tm3(CAG-EYFP)Hze /J) The Jackson Laboratory Strain# 007903; RRID: IMSR_JAX: 007903 GLP1r-cre Dr. Fiona Gribble (University of Cambridge) N/A TH-Cre Dr. Viviana Gradinaru (Caltech) N/A Software and Algorithms MATLAB R2016a/2017b MathWorks N/A Prism 7/8 GraphPad N/A Photoshop CS6/CC Adobe N/A Illustrator CS5/CC Adobe N/A LASX Leica N/A Office 2016 Microsoft Open in a separate window KEY RESOURCES TABLE Thirst neurons in the brain receive temporally distinct multiple satiation signals Liquid gulping and gut osmolality signals transmit thirst satiation Specific GABAergic neurons in the subfornical organ (SFO) mediate satiation signals from the gut Drinking-induced dopamine release is independent of thirst satiation signals

Techniques: Plasmid Preparation, Injection, Recombinant, Knock-In, Software