rabbit polyclonal primary antiserum directed against inos  (Santa Cruz Biotechnology)

Journal: Cells

Article Title: IL-6 Is Not Absolutely Essential for the Development of a TH17 Immune Response after an Aerosol Infection with Mycobacterium tuberculosis H37rv

doi: 10.3390/cells10010009

Figure Lengend Snippet: The TH1 immune response and macrophage effector functions in IL-6 −/− mice during experimental TB. C57BL/6 (grey symbols) and IL-6 −/− (red symbols) mice were infected with approximately 100 CFU Mtb via the aerosol route. At the indicated time points, lungs were removed and further processed. ( a ) The relative amount of IFNγ-producing CD4 + CD44 + CD90.2 + T cells was determined in single cell suspensions of lungs by flow cytometry after restimulation with anti-CD3/CD28. Representative density plot of IFNγ-producing CD4 + CD44 + CD90.2 + T cells on day 42 and IFNγ-producing CD4 + CD44 + CD90.2 + T cells during the course of infection are shown. ( b ) CD4 + T cells were purified from lung cell suspensions by MACS and restimulated with ESAT-6 1-20 . The frequency of IFN γ-producing cells was determined by ELISPOT. ( c ) Gene expression of Nos2 and Lrg47 was quantified by real-time RT-PCR based on gene expression in uninfected mice. ( d ) Representative immunohistochemical staining of NOS2 in lung sections of experimental mice 42 days of infection. ( e ) At the indicated time points, bacterial loads in the lungs were determined. Mean values with standard deviations of 5 mice per group are shown. One representative experiment of two is shown. Data were statistically analyzed using the Mann–Whitney test defining differences between C57BL/6 and IL-6 −/− mice as significant (**, p ≤ 0.01).

Article Snippet: For the immunohistochemical detection of NOS2, sections were stained with a polyclonal rabbit anti-mouse NOS2 antiserum (Biomol, Hamburg, Germany) and examined microscopically.

Techniques: Infection, Aerosol, Flow Cytometry, Purification, Enzyme-linked Immunospot, Gene Expression, Quantitative RT-PCR, Immunohistochemical staining, Staining, MANN-WHITNEY

Journal: Cells

Article Title: IL-6 Is Not Absolutely Essential for the Development of a TH17 Immune Response after an Aerosol Infection with Mycobacterium tuberculosis H37rv

doi: 10.3390/cells10010009

Figure Lengend Snippet: The TH1 immune response in CD4 cre ; gp130 loxP/loxP mice during experimental TB. gp130 loxP/loxP (grey symbols) and CD4 cre ; gp130 loxP/loxP mice (green symbols) were infected with approximately 100 CFU Mtb via the aerosol route. At the indicated time points, lungs were removed and further processed. ( a ) The relative amount of IFNγ-producing CD4 + CD44 + CD90.2 + T cells was determined in single cell suspensions of lungs by flow cytometry after restimulation with anti-CD3/CD28. Representative density plots of IFNγ-producing CD4 + CD44 + CD90.2 + T cells on day 42 and IFNγ-producing CD4 + CD44 + CD90.2 + T cells during the course of infection are shown. ( b ) CD4 + T cells were purified from lung cell suspensions by MACS and restimulated with ESAT-6 1-20 . The frequency of IFNγ-producing cells was determined by ELISPOT. ( c , d ) After reverse transcription of isolated lung RNA, gene expression of ( c ) Nos2 and Lrg47 was quantified by real-time RT-PCR based on gene expression in uninfected mice. ( d ) Representative immunohistochemical staining of NOS2 in lung section of experimental mice 42 days of infection. ( e ) At the indicated time points, bacterial loads in the lungs were determined. Mean values with standard deviations of 5 mice per group are shown. One representative experiment of two is shown. Data were statistically analyzed using the Mann–Whitney test defining differences between cre-negative littermates and CD4 cre ; gp130 loxP/loxP mice as significant (*, p ≤ 0.05; **, p ≤ 0.01).

Article Snippet: For the immunohistochemical detection of NOS2, sections were stained with a polyclonal rabbit anti-mouse NOS2 antiserum (Biomol, Hamburg, Germany) and examined microscopically.

Techniques: Infection, Aerosol, Flow Cytometry, Purification, Enzyme-linked Immunospot, Reverse Transcription, Isolation, Gene Expression, Quantitative RT-PCR, Immunohistochemical staining, Staining, MANN-WHITNEY

Journal: American Journal of Reproductive Immunology

Article Title: Erythropoietin prevents LPS‐induced preterm birth and increases offspring survival

doi: 10.1111/aji.13283

Figure Lengend Snippet: Effect of EPO and LPS on uterine phosphorylated‐NF‐κB‐65 and iNOS protein levels. Uterine strips were collected, phosphorylated‐NF‐κB‐65 and iNOS protein levels were assessed at 12 hours after the first LPS or vehicle solution injection. The levels of phosphorylated‐NF‐κB‐65 and iNOS were determined by Western blots. The levels of phosphorylated‐NF‐κB‐65 (A) and iNOS (B) were determined. Values are presented as mean ± SD. N = 5. * P < .01 vs the control. * P < .05, ** P < .01 vs LPS group

Article Snippet: The membranes were placed in the blocking buffer for 1 hour at room temperature (RT) and incubated with a primary rabbit antiserum against iNOS (1:1000; CST) or phosphorylated‐NF‐κB‐65 (1:1000; CST) overnight at 4°C.

Techniques: Injection, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Single Administration of Melatonin Modulates the Nitroxidergic System at the Peripheral Level and Reduces Thermal Nociceptive Hypersensitivity in Neuropathic Rats

doi: 10.3390/ijms18102143

Figure Lengend Snippet: Constitutive (neuronal) isoform (nNOS) and inducible isoform (iNOS) evaluation in dorsal root ganglia (DRG). ( a ) Quantitative evaluation of nNOS immunopositivity in DRG (small size neurons − diameter < 30 µm) as IOD (integrated optical density) in the experimental animals: sham-operated rats treated with 1% ethanol in saline (vehicle for melatonin) (SHAM + V); sham-operated rats treated with melatonin (10 mg/kg) (SHAM + MEL10); CCI rats treated with 1% ethanol in saline (vehicle for melatonin) (CCI + V); CCI rats treated with melatonin (5 mg/kg) (CCI + MEL5); CCI animals treated with melatonin (10 mg/kg) (CCI + MEL10). Data represent means ± SEM, ° p < 0.05 vs. SHAM + V, # p < 0.05 vs. CCI + V, § p < 0.05 vs. CCI + MEL5. ( b ) Quantitative evaluation of iNOS immunopositivity in dorsal root ganglia (DRG) (small size neurons − diameter < 30 µm) as IOD (integrated optical density) in the experimental animals: sham-operated rats treated with 1% ethanol in saline (vehicle for melatonin) (SHAM + V); sham-operated rats treated with melatonin (10 mg/kg) (SHAM + MEL10); CCI rats treated with 1% ethanol in saline (vehicle for melatonin) (CCI + V); CCI rats treated with melatonin (5 mg/kg) (CCI + MEL5); CCI animals treated with melatonin (10 mg/kg) (CCI + MEL10). Data represent means ± SEM, ° p < 0.05 vs. SHAM + V, # p < 0.05 vs. CCI + V.

Article Snippet: Briefly, the sections were incubated firstly in normal goat serum (10% in TBS plus 0.1% Triton X-100) for 60 min, and then in rabbit polyclonal primary antiserum directed against nNOS and iNOS (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted, respectively, at 1:200 and 1:500 in TBS containing 3% normal goat serum and 0.1% Triton X-100, for 24 h at 4 °C.

Techniques: Saline

Journal: International Journal of Molecular Sciences

Article Title: Single Administration of Melatonin Modulates the Nitroxidergic System at the Peripheral Level and Reduces Thermal Nociceptive Hypersensitivity in Neuropathic Rats

doi: 10.3390/ijms18102143

Figure Lengend Snippet: nNOS and iNOS evaluation in plantar skin. ( a ) Quantitative evaluation of nNOS immunopositivity in the epidermis of the right hind-paw plantar skin as IOD (integrated optical density) in the experimental animals: sham-operated rats treated with 1% ethanol in saline (vehicle for melatonin) (SHAM + V); sham-operated rats treated with melatonin 10 mg/kg (SHAM + MEL10); CCI rats treated with 1% ethanol in saline (vehicle for melatonin) (CCI + V); CCI rats treated with melatonin (5 mg/kg) (CCI + MEL5); CCI rats treated with melatonin (10 mg/kg) (CCI + MEL10). Data represent means ± SEM, ° p < 0.05 vs. SHAM + V, * p < 0.05 vs. SHAM + MEL10, # p < 0.05 vs. CCI + V. ( b ) Quantitative evaluation of iNOS immunopositivity in the epidermis of the right hind-paw plantar skin as IOD (integrated optical density) in the experimental animals: sham-operated rats treated with 1% ethanol in saline (vehicle for melatonin) (SHAM + V); sham-operated rats treated with melatonin 10 mg/kg (SHAM + MEL10); CCI rats treated with 1% ethanol in saline (vehicle for melatonin) (CCI + V); CCI rats treated with melatonin (5 mg/kg) (CCI + MEL5); CCI rats treated with melatonin (10 mg/kg) (CCI + MEL10). Data represent means ± SEM, ° p < 0.05 vs. SHAM + V, # p < 0.05 vs. CCI + V, § p < 0.05 vs. CCI + MEL5.

Article Snippet: Briefly, the sections were incubated firstly in normal goat serum (10% in TBS plus 0.1% Triton X-100) for 60 min, and then in rabbit polyclonal primary antiserum directed against nNOS and iNOS (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted, respectively, at 1:200 and 1:500 in TBS containing 3% normal goat serum and 0.1% Triton X-100, for 24 h at 4 °C.

Techniques: Saline

Journal: International Journal of Molecular Sciences

Article Title: Single Administration of Melatonin Modulates the Nitroxidergic System at the Peripheral Level and Reduces Thermal Nociceptive Hypersensitivity in Neuropathic Rats

doi: 10.3390/ijms18102143

Figure Lengend Snippet: Schematic diagram of potential melatonin pathways at the skin and DRG levels. The diagram represents the potential pathways undergoing the beneficial effect of melatonin in neuropathic pain conditions at skin and DRG levels. Melatonin is highly lipid soluble, and readily crosses the plasma membrane to enter the cell; it has access to cytosolic, mitochondrial, and nuclear compartments. Through its radical scavenging action, it protects the cells from free radicals, such as nitric oxide (NO) and reactive oxygen species (ROS), and decreases the neurotransmitters/neuromodulators production, which leads to the nerve fibres excitation. So, the beneficial effect of melatonin at the skin level could also affect the sensory nerve terminals influencing the nociceptive transmission. Melatonin may also inhibit directly the catalytic activity of nitric oxide synthase (NOS) and modulate NOS expression. In particular, iNOS expression is reduced through the inhibition of the transcription factor nuclear factor-kappa B (NF-κB), both at the DRG and in keratinocytes. Regarding nNOS, at the skin level, we presume that its increased expression in sham-operated animals treated with melatonin (10 mg/kg) may be probably due to the melatonin interaction with RORα receptor, even if it has not yet been identified in the rat, and only in the mouse. At the DRG level, nNOS upregulation seems to represent a general response of neuronal cells to stress, including nerve injuries, as in chronic constriction injury model (CCI). Moreover, melatonin may reduce the intracellular calcium rise by inhibiting, for example, high voltage activated calcium channels (HVACC), and thereby inhibiting also nNOS activation.

Article Snippet: Briefly, the sections were incubated firstly in normal goat serum (10% in TBS plus 0.1% Triton X-100) for 60 min, and then in rabbit polyclonal primary antiserum directed against nNOS and iNOS (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted, respectively, at 1:200 and 1:500 in TBS containing 3% normal goat serum and 0.1% Triton X-100, for 24 h at 4 °C.

Techniques: Clinical Proteomics, Membrane, Transmission Assay, Activity Assay, Expressing, Inhibition, Activation Assay