goat polyclonal antibody against human tnfα  (R&D Systems)

Journal: Microorganisms

Article Title: Adenoviral Vector Codifying for TNF as a Co-Adjuvant Therapy against Multi-Drug-Resistant Tuberculosis

doi: 10.3390/microorganisms11122934

Figure Lengend Snippet: Gene expression of pro-inflammatory cytokines in the different experimental groups. Gene expression quantification by qPCR using the 2^ (−ΔCt) method relative to Rplp0 (housekeeping gene) of ( A ) Ifng , ( B ) Tnf and ( C ) Nos2 at day 14, 28 and 60 of treatment with AdTNF, AdGFP or SS. At day 60 of treatment, a significant increase in gene expression was induced by AdTNF treatment. Asterisks represent statistical significance (**** p < 0.0001, two-way ANOVA).

Article Snippet: Cytokine detection was performed with goat anti-mouse polyclonal antibodies against TNF, IFN-γ and iNOS (Santa Cruz Biotechnology, Santa Cruz, CA, USA), secondary anti-goat antibody-HRP (Goat-on-Rodent HRP-Polymer, Biocare Medical); finally, slides were revealed with diaminobenzidine/H 2 O 2 and contrasted with hematoxylin stain.

Techniques: Gene Expression

Journal: Microorganisms

Article Title: Adenoviral Vector Codifying for TNF as a Co-Adjuvant Therapy against Multi-Drug-Resistant Tuberculosis

doi: 10.3390/microorganisms11122934

Figure Lengend Snippet: Percentage of immunostained cells to pro-inflammatory cytokines and iNOS in pneumonic areas of the different experimental groups. Percentage of immunostained cells to ( A ) IFN-γ, ( B ) TNF and ( C ) iNOS in the pneumonic areas of mice infected with MDR-TB after the indicated days of treatment with one dose of AdTNF, AdGFP or SS administered by intratracheal route. AdTNF induced a significant increment of immunostained cells in all the evaluated time points. Asterisks represent statistical significance (**** p < 0.0001, two-way ANOVA). ( D ) Representative microphags (40×) of immunostaining detection (brown cytoplasm peroxidase staining) of the indicated cytokine and iNOS in pneunmonic areas comparing AdTNF and AdGFP.

Article Snippet: Cytokine detection was performed with goat anti-mouse polyclonal antibodies against TNF, IFN-γ and iNOS (Santa Cruz Biotechnology, Santa Cruz, CA, USA), secondary anti-goat antibody-HRP (Goat-on-Rodent HRP-Polymer, Biocare Medical); finally, slides were revealed with diaminobenzidine/H 2 O 2 and contrasted with hematoxylin stain.

Techniques: Infection, Immunostaining, Staining

Journal: Glia

Article Title: Tumor necrosis factor receptor-1 is selectively sequestered into Schwann cell extracellular vesicles where it functions as a TNFα decoy

doi: 10.1002/glia.24098

Figure Lengend Snippet: TNFR1 is selectively sequestered into SC EVs. (a) SC EVs isolated by UC from conditioned media (CM) of primary cultured SCs were analyzed by NTA and had an average particle size of 153 ± 2 nm; n = 3 independent experiments. (b) Transmission electron microscopy (TEM) using negative staining of EVs. Note large crescent shaped particles and smaller particles. Scale bar, 200 nm. (c) Immunoblot analysis of whole SC lysates (SC-L) and SC-derived EVs to detect the exosome biomarkers, Flotillin-1, TSG101, CD9, ALIX, and CD81. GM130 is a Golgi biomarker not found in exosomes. (d) Immunoblot analysis to detect the SC biomarkers, p75NTR and myelin protein zero, P0, and non-myelinating marker, GFAP. Images represent n = 3–5 independent experiments. (e) TNFα receptors, TNFR1 (55-kDa) and TNFR2 (65-kDa), and P0 in extracts of SCs cultured in complete medium (SC-L1) or in DMEM with 10% FBS depleted of EVs (SC-L2), in SC-EVs, and in bone marrow derived macrophages (BMDMs) (2 μg/lane) were determined by immunoblot analysis. (f) Immunoblot of TNFR1 levels in EVs derived from SCs treated with and without TNFα. TSG101 (44-kDa) shows load control in cells and presence in EVs. (g) Quantification of TNFR1 levels in SC EV immunoblots (2 μg). Data are expressed as mean ± SEM; (n = 4–5/group)

Article Snippet: Incubations with primary goat polyclonal antibody against human TNFα (1:1000; R&D Systems, AF-510-NA) were performed overnight at 4°C.

Techniques: Isolation, Cell Culture, Transmission Assay, Electron Microscopy, Negative Staining, Western Blot, Derivative Assay, Biomarker Discovery, Marker, Control

Journal: Glia

Article Title: Tumor necrosis factor receptor-1 is selectively sequestered into Schwann cell extracellular vesicles where it functions as a TNFα decoy

doi: 10.1002/glia.24098

Figure Lengend Snippet: TNFR1 is expressed on the surface of SC EVs and binds TNFα in vitro. (a) Representative dot blot of non-permeabilized SC EVs (0–2.5 μg) probed with anti-TNFR1 antibody (n = 3 independent blots). (b) Standard curve showing recombinant soluble TNFR1 (28-kDa) and two representative SC EV samples (1 μg/lane). (c) Quantification of the TNFR1 copy number based on the TNFR1 standard curve, NTA and BCA analysis of six independent EV preparations. Data are expressed as the mean ± SEM. (d) Representative immunoblot of TNFα treated with the crosslinker, BS3 (+), or with vehicle (−) for the indicated times at 37°C. (e) Representative immunoblot of SC EVs, SC EV + TNFα and TNFα alone treated with BS3. Note the high molecular mass band in sample of EVs plus TNFα, compared with TNFα alone. (f) Dot blot of four independent SC EV preparations (EV1–4; 1 μg) incubated with or without biotinylated TNFα and probed with S-HRP

Article Snippet: Incubations with primary goat polyclonal antibody against human TNFα (1:1000; R&D Systems, AF-510-NA) were performed overnight at 4°C.

Techniques: In Vitro, Dot Blot, Recombinant, Western Blot, Incubation

Journal: Glia

Article Title: Tumor necrosis factor receptor-1 is selectively sequestered into Schwann cell extracellular vesicles where it functions as a TNFα decoy

doi: 10.1002/glia.24098

Figure Lengend Snippet: SC EV TNFR1 functions as a decoy by binding TNFα and inhibiting its association with cells. (a) Representative IF microscopy images identifying cell associated TNFα (red) and SC nuclei with DAPI (blue) in non-permeabilized SCs. Primary SCs were treated with TNFα (1 nM) in the presence or absence of SC EVs (5 μg) for 1 h and fixed in 4% paraformaldehyde. Scale bar, 50 μm. (b) Immunoblot of TNFα in SC extracts after incubating SCs with TNFα in the presence or absence of SC EVs (5 μg) for 1 h. Membranes were re-probed with GAPDH as a loading control (lower panel). (c) Densitometry analysis showing SC EV-induced reductions in cell-associated TNFα, standardized to the loading control (mean ± SEM; n = 3/group; *p < .05 using a one-way ANOVA and a Tukey’s post hoc test). (d) Immunoblot analysis identifying TNFα (1 nM) in SC CM after incubating SCs with TNFα in the presence or absence of SC EV (5 μg) for 1 h

Article Snippet: Incubations with primary goat polyclonal antibody against human TNFα (1:1000; R&D Systems, AF-510-NA) were performed overnight at 4°C.

Techniques: Binding Assay, Microscopy, Western Blot, Control

Journal: Glia

Article Title: Tumor necrosis factor receptor-1 is selectively sequestered into Schwann cell extracellular vesicles where it functions as a TNFα decoy

doi: 10.1002/glia.24098

Figure Lengend Snippet: SC EVs attenuate TNFα-induced p38 MAPK activation in vitro. (a) Representative immunoblot showing activation of p38 MAPK (P-p38) by TNFα (0.5 nM) and the effects of SC EVs (10 μg), when added simultaneously. Membranes were re-probed for total p38 MAPK (T-p38) as a loading control. Equal amounts of protein extracts (20 μg) were loaded into each lane. (b) Densitometry analysis was performed to determine the relative level of P-p38 standardized against T-p38 (mean ± SEM; n = 10 independent experiments, **p < .01, vehicle versus TNFα; **p < .01, TNFα versus SC EV using a one-way ANOVA with a Tukey’s post hoc test). (c) Representative immunoblot showing that TNFα (0.5 nM) activates p38 MAPK and that activation is not blocked by two concentrations of pre-incubated TNFR1-neutralizing antibody. (d) Immunoblot analysis of phospho-p38 MAPK in response to TNFα in the presence or absence of SC EVs. SC EVs were added to cultures together with TNFR1 neutralizing antibody (TNFR1 Ab; 2.5 or 10 μg) as indicated. The blot is representative of three independent experiments. (e) Immunoblot analysis of TNFR1 in pellet and supernatant of SC EVs treated with TACE/ADAM17 (20 or 80 nM). TSG101, an EV biomarker is identified in the pellet. Equal amounts of protein extracts (12 μg) were loaded into each lane. (f) Immunoblot analysis of TNFR1 in control SC EVs and TACE-treated SC EVs. TSG101 serves as a loading control. (g) Representative immunoblot showing activation of p38 MAPK by TNFα (0.5 nM) and the effects of independent preparations of TACE-EVs (10 μg/ml). Membranes were re-probed for T-p38 as a loading control. Equal amounts of protein extracts (20 μg) were loaded into each lane. (h) Densitometry analysis was performed to determine the relative level of P-p38 standardized against T-p38 (mean ± SEM; n = 6 independent experiments, ***p < .001, vehicle versus TNFα; ***p < .001 vehicle versus TNFα + TACE-EV; p = .997, n.s. TNFα versus TACE-EV using a one-way ANOVA with a Tukey’s post hoc test)

Article Snippet: Incubations with primary goat polyclonal antibody against human TNFα (1:1000; R&D Systems, AF-510-NA) were performed overnight at 4°C.

Techniques: Activation Assay, In Vitro, Western Blot, Control, Incubation, Biomarker Discovery

Journal: Glia

Article Title: Tumor necrosis factor receptor-1 is selectively sequestered into Schwann cell extracellular vesicles where it functions as a TNFα decoy

doi: 10.1002/glia.24098

Figure Lengend Snippet: TNFR2 mediates TNFα induced P38 MAPK pathway in cultured SCs. (a) RT-qPCR analysis of TNFR2 mRNA after transfection with TNFR2 siRNA for 48 h. Data are presented as mean ± SEM; n = 4 independent experiments, *p < .05 using a t test. (b) Representative immunoblot analysis of phospho-p-38 MAPK (P-p38) that is dose-dependently increased by TNFα (8 min) in NTC cells, but not in cells transfected with TNFR2-specific siRNA (n = 3). (c) RT-qPCR analysis of TNFR1 mRNA after TNFR2 siRNA transfection for 48 h. Data are presented as mean ± SEM; n = 6 independent experiments, n.s. using a t test. (d) Representative immunoblot analysis of P-p38 after TNFα (0.5 nM) stimulation for 8 min in SCs treated dose-dependently with neutralizing anti-TNFR2 antibody (0–8 μg) or IgG control (8 μg)

Article Snippet: Incubations with primary goat polyclonal antibody against human TNFα (1:1000; R&D Systems, AF-510-NA) were performed overnight at 4°C.

Techniques: Cell Culture, Quantitative RT-PCR, Transfection, Western Blot, Control

Journal: Glia

Article Title: Tumor necrosis factor receptor-1 is selectively sequestered into Schwann cell extracellular vesicles where it functions as a TNFα decoy

doi: 10.1002/glia.24098

Figure Lengend Snippet: SC EVs promote cell morphology changes in SCs following TNFα exposure. (a) Immunofluorescence of S100β in primary cultured SCs treated with vehicle, TNFα (0.5 nM), or TNFα + SC EVs (10 μg) for 1.5 h. Dapi (blue) labels nuclei. Scale bar, 30 μm. (b) Quantitative analysis of S100β immunofluorescence and Dapi. The N/C ratio of SCs treated with TNFα or TNFα in the presence of SC EVs was calculated (mean ± SEM.; n = 30–40 cells across three biological replicates, ***p < 0.001, one-way ANOVA and Tukey’s post hoc test)

Article Snippet: Incubations with primary goat polyclonal antibody against human TNFα (1:1000; R&D Systems, AF-510-NA) were performed overnight at 4°C.

Techniques: Immunofluorescence, Cell Culture

Journal: Glia

Article Title: Tumor necrosis factor receptor-1 is selectively sequestered into Schwann cell extracellular vesicles where it functions as a TNFα decoy

doi: 10.1002/glia.24098

Figure Lengend Snippet: Effect of SC EVs on TNFα-mediated cytokine production and cell death. (a) Array analyses of cytokines in SC cell lysates after incubating SCs with TNFα or TNFα plus SC EVs for 3 h. Duplicate dots representing individual cytokines that were increased in abundance by greater than 1.3-fold by EVs, or decreased by greater than 50% by EVs, are indicated by colored boxes. (b) Densitometric analysis of cytokines regulated by SC EVs in SCs treated with TNFα. Data are expressed as the fold-change in cells treated with TNFα + SC EVs versus TNFα alone. (c) Cell death Elisa™ results are shown. TNFα (0.5 nM) treatment for 18 h increased SC death by sevenfold compared with control conditions (10% EV-depleted FBS containing media) and > twofold compared with vehicle control (1% EV-depleted FBS containing media). The increase in cell death was attenuated by co-treatment with SC EVs (0.5 or 1.0 μg) (mean ± SEM; n = 3 independent experiments, *p < .05 using a one-way ANOVA with a Tukey’s post hoc test). (d) Dose-dependent effects of SC EVs on SCs treated with TNFα was determined by trypan blue exclusion assay (mean ± SEM; n = 3 independent experiments, *p < .05 using a one-way ANOVA with a Tukey’s post hoc test)

Article Snippet: Incubations with primary goat polyclonal antibody against human TNFα (1:1000; R&D Systems, AF-510-NA) were performed overnight at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Trypan Blue Exclusion Assay

Journal: Glia

Article Title: Tumor necrosis factor receptor-1 is selectively sequestered into Schwann cell extracellular vesicles where it functions as a TNFα decoy

doi: 10.1002/glia.24098

Figure Lengend Snippet: SC EVs block the effects of TNFα on sciatic nerve morphology and pain in vivo. (a) Representative immunoblot showing activation of p38 MAPK in naïve sciatic nerve (no injury), in sciatic nerve 15 min after injection of vehicle, TNFα (1 ng), or TNFα (1 ng) plus SC EVs (0.5 μg). (b) Densitometry analysis was performed to determine the relative levels of P-p38 MAPK standardized to the loading control (mean ± SEM; n = 4/group; *p < .05 compared with control group by a Kruskal-Wallis test and a Dunn’s multiple comparisons test). (c) Sciatic nerves were injected with TNFα (0.5 ng), TNFα plus SC EVs (1.8 μg), or vehicle as indicated. Images of individual (SN1, SN2, SN3) rat sciatic nerves (H&E-stained transverse sections) harvested immediately distal to the injection site after 3 days. Note edema induced by TNFα alone is not observed in the two other groups (magnification ×100; scale bar 100 μm). (d) Rats were baseline tested with von Frey hairs for 3 days prior to a one-time injection with vehicle (0.01% BSA), TNFα (0.5 ng), or TNFα + SC EV (1.8 μg). PWTs were determined on day 3 (mean ± SEM; n = 4–7/group; *p < .05 as compared with the corresponding basal PWTs and between TNFα and TNFα + SC EV using a repeated measures ANOVA with a Bonferroni post hoc test)

Article Snippet: Incubations with primary goat polyclonal antibody against human TNFα (1:1000; R&D Systems, AF-510-NA) were performed overnight at 4°C.

Techniques: Blocking Assay, In Vivo, Western Blot, Activation Assay, Injection, Control, Staining

Journal: Scientific Reports

Article Title: Effect of amniotic fluid stem cell transplantation on the recovery of bladder dysfunction in spinal cord-injured rats

doi: 10.1038/s41598-020-67163-7

Figure Lengend Snippet: Immunostaining for IL-6 and TNF-alpha in the bladder sections of sham rats (Sham) and spinal cord-injured (SCI) rats after phosphate-buffered saline (SCI + PBS), HEK293 cells (SCI + HEK293) and hAFSC (SCI + hAFSC) transplantation. Compared to that in the sham group, the expression of IL-6 and TNF-alpha in bladder tissue was significantly increased in the SCI + PBS and SCI + HEK293 groups. hAFSC transplantation recovered IL-6 and TNF-alpha to near sham levels at day 7, but less recovery was observed at day 28. Bar in the sham panel = 40 μm. *P < 0.05 vs. sham group. N = 6 at each time point. HEK293 = human embryonic kidney 293 cells. IL-6 = interleukin-6. TNF-alpha = tumor necrosis factor-alpha.

Article Snippet: Sections were incubated for 18–20 h at 4 °C with rabbit polyclonal antibodies against TGF-β1 (1:50, OriGene Technologies, Inc., Rockville, MD, USA), β3-adrenoceptor (1:750, Millipore, Temecula, CA), M2 (1:1,000, Millipore, Temecula, CA, USA), M3 (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and IL-6 (1:200, GeneTex, Irvine, CA, USA), mouse monoclonal antibodies against IGF-1 (1:500, Abcam, Cambridge, MA, USA) and BDNF (1:750, OriGene Technologies, Inc. Rockville, MD, USA) and goat polyclonal antibody against TNF-alpha (1:200, R&D Systems, Minneapolis, MN, USA).

Techniques: Immunostaining, Saline, Transplantation Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Beneficial Effects of Melatonin on Apolipoprotein-E Knockout Mice by Morphological and 18 F-FDG PET/CT Assessments

doi: 10.3390/ijms21082920

Figure Lengend Snippet: Immunofluorescence characterization of macrophage cytokine production. Labelling of the aorta’s wall with TNF-α ( a , b ) and TGF-β ( d , e ) antibodies. Each panel shows a full-field image at 400× (scale bars: 20 µm). Immunofluorescence photomicrograph of control mice showed a moderate TNF-α expression at the subendothelial and adventitia levels, confirming that these mice were characterized by an infiltrate of macrophages predominantly polarized in the M1 direction ( a ). In contrast, the immunofluorescence photomicrograph of ApoE -/- mice treated with melatonin highlights, for the same M1 marker, an absent/very weak positivity ( b ). Immunofluorescence photomicrograph also showed an absent expression of TGF-β in the aorta of the control mice ( d ). Differently, the immunofluorescence photomicrograph of MLT-treated mice showed a moderate TGF-β expression in the tunica adventitia ( e ). Graphs summarize, as arbitrary units, the immunomorphometrical analyses of TNF-α ( c ) and TGF-β ( f ) obtained, for both pro-inflammatory markers, evaluating fifteen non-overlapping fields with the same area for each experimental animal, respectively. For both TNF-α ( g ) and TGF-β ( h ), sections without primary antibody and in the presence of isotype matched immunoglobulins G served as negative immunofluorescence controls. Statistical analyses comparing multiple continuous outcomes were performed using one-way analyses of variance test corrected by Bonferroni for immunomorphometrical evaluations. Continuous variables are summarized as means ± standard deviation. Error bars represent the 95% confidence interval around the mean; * indicates the level of significance, p ≤ 0.05; red arrows indicate the positive staining for TNF-α; green arrows indicate the positive staining for TGF-β; I, tunica intima; M, tunica media; A, tunica adventitia; CTR, control group; MLT, mice treated with melatonin; AU, arbitrary units.

Article Snippet: Alternate paraffin sections were dewaxed, rehydrated, and incubated in 3% hydrogen peroxide for 30 min. Then, after the blocking step in 3% bovine serum albumin solution for 1 h, the sections were incubated 1 h at 37 °C and 30 min at room temperature with the following primary antibodies: rat monoclonal antibody against CD68 (diluted 1:100; Abcam, Cambridge, UK); rabbit polyclonal antibody against CD163 (diluted 1:50; Abcam, Cambridge, UK); goat polyclonal antibody against TNF-α (diluted 1:200 Santa Cruz Biotechnology Inc., Dallas, TX, USA) [ ]; mouse monoclonal antibody against TGF-β (diluted 1:150; Santa Cruz Biotechnology Inc., Dallas, TX, USA) [ ] and simultaneously with mouse monoclonal antibody against ICAM-1 (diluted 1:200; Santa Cruz Biotechnology Inc., Dallas, TX, USA) and rabbit polyclonal antibody against VCAM-1 (diluted 1:200; Santa Cruz Biotechnology Inc., Dallas, TX, USA).

Techniques: Immunofluorescence, Expressing, Marker, Standard Deviation, Staining