mTNF-a Aff Pur PAb (100 ug)

Detection of Human TNF‑ alpha by Western Blot. Western blot shows conditioned media from CHO Chinese hamster ovary cell line either mock transfected or transfected with human TNF- alpha. PVDF membrane was probed with 2 µg/mL of Goat Anti-Human/Mouse TNF-a Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-410-NA) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (HAF017). Specific bands were detected for TNF-a at approximately 14 and 17 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Mouse TNF‑ alpha by Western Blot. Western blot shows lysates of RAW 264.7 mouse monocyte/macrophage cell line untreated (-) or treated (+) with 10 µg/mL LPS for 4 hours. PVDF membrane was probed with 2 µg/mL of Goat Anti-Human/Mouse TNF-a Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-410-NA) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (HAF017). Specific bands were detected for TNF-a at approximately 14 and 17 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

TNF‑ alpha in RAW 264.7 Mouse Cell Line. TNF-a was detected in immersion fixed RAW 264.7 mouse monocyte/ macrophage cell line treated with LPS using Human/Mouse TNF-a Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-410-NA) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; NL001) and counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

TNF‑ alpha in Mouse T Cells. TNF‑ alpha was detected in immersion fixed activated mouse T Cells using 15 µg/mL Human/Mouse TNF‑ alpha Antigen Affinity-purified Polyclonal Antibody (Catalog # AF‑410‑NA) for 3 hours at room temperature. Cells were stained (red) and counterstained (green). View our protocol for Fluorescent ICC Staining of Non-adherent Cells.

TNF-alpha in Mouse Spleen Tissue. TNF‑ alpha was detected in immersion fixed paraffin-embedded sections of mouse spleen tissue using Goat Anti-Human/Mouse TNF‑ alpha Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-410-NA) at 3 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (VC004). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to lymphocytes. Staining was performed using our IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Cytotoxicity Induced by TNF‑ alpha and Neutralization by Mouse TNF‑ alpha Antibody. Recombinant Mouse TNF-a (410-MT) induces cytotoxicity in the the L-929 mouse fibroblast cell line in a dose-dependent manner (orange line). Cytotoxicity elicited by Recombinant Mouse TNF-a (0.1 ng/mL) is neutralized (green line) by increasing concentrations of Human/Mouse TNF-a Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-410-NA). The ND₅₀ is typically 1.5-10 ng/mL in the presence of the metabolic inhibitor actinomycin D.

Detection of TNF‑ alpha in Human Spleen. TNF‑ alpha was detected in immersion fixed paraffin-embedded sections of Human Spleen using Goat Anti-Human/Mouse TNF‑ alpha Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-410-NA) at 5 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC004). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using VisUCyte Antigen Retrieval Reagent-Basic (Catalog # VCTS021). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm in lymphocytes. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Detection of Mouse TNF-alpha by Western Blot WNT-5A induces a proinflammatory transformation in mouse microglia. (A, B) iNOS, COX-2 and TNF alpha were detected by immunoblotting in lysates from mouse primary microglia after WNT-5A stimulation (ctrl, 300 ng/ml, 6 hours). (B’) shows TNF alpha levels in the supernatant of primary microglia under ctrl conditions and upon WNT-5A stimulation (ctrl, 300 ng/ml, 24 hours; n = 4). At least three experiments are summarized in the bar graphs. Data are normalized to ctrl. Error bars give s.e.m. (C) Microglial proliferation was assessed by an MTT assay monitoring mitochondrial activity, which is proportional to cell number [see Additional file 1: Figure S5]. Stimulation with WNT-5A (300 ng/ml, 24 hours) increased MTT, which was blocked by PTX (100 ng/ml, overnight) or the MEK1/2 (10 μM) inhibitor, SL327. (D). Experiments were done in triplicate and data from three independent experiments are shown. *, P < 0.01; ***, P < 0.001: Error bars show s.e.m.. (E) Cell tracker (red)-stained primary microglia were seeded on top of a collagen matrix in 35 mm glass bottom dishes. One day after ctrl or 300 ng/ml WNT-5A stimulation, invasion was observed by confocal microscopy and Z-stacking using a Zeiss LSM710 microscope and subsequent analysis with the Bitplane Imaris software. The size of the collagen cube shown is 1,000 (Z) x 1,400 (Y) x 1,400 (X) μm. Three invasion experiments in the absence and presence of the MEK1/2 inhibitor SL327 were quantified. Data are presented in a bar graph (F). *, P < 0.05; error bars show s.e.m.. (G) cDNA of ctrl stimulated (−) and WNT-5A stimulated (+) primary microglia was analyzed by QPCR for expression of inflammatory genes. At least three independent experiments are summarized. Gene expression is normalized to the housekeeping gene GAPDH and expressed as arbitrary units (2-delta ct). *, P < 0.05; **, P < 0.01. COX-2, cyclooxygenase 2; iNOS, inducible nitric oxide synthase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; n, number; s.e.m., standard error of the mean. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/22647544), licensed under a CC-BY license. Not internally tested by R&D Systems.