TNF alpha anticorps

N° du produit ABIN2689935

Détail du produit anti-TNF alpha anticorps

Antigène: TNF alpha (Tumor Necrosis Factor alpha (TNF alpha))

Reactivité: Humain

Hôte: Souris

Clonalité: Monoclonal

Conjugué: Cet anticorp TNF alpha est non-conjugé

Application: Immunocytochemistry (ICC)

Marque: BD Pharmingen™

Attributs du produit: The MAb11 antibody reacts with human tumor necrosis factor (TNF, also known as TNF-α) protein. TNF is an efficient paracrine and endocrine mediator of inflammatory and immune functions. It regulates the growth and differentiation of a variety of cell types. TNF is cytotoxic for transformed cells when in conjunction with IFN-γ. It is secreted by activated monocytes/macrophages and other cells such as B cells, T cells and fibroblasts. The immunogen used to generate the MAb11 hybridoma was recombinant human TNF. The use of the MAb11 antibody has been reported to cross-react with TNF of rhesus monkey. This antibody is routinely tested by immunocytochemical staining. Other applications were tested during antibody development only or reported in the literature. PBMC were isolated from human peripheral blood by density gradient centrifugation and were cultured for 2 days with plate bound anti-human CD3 and soluble anti-human CD28 in the presence of human recombinant IL-2 and human recombinant IL-4. The cells were subsequently harvested, washed and recultured with human recombinant IL-2 and human recombinant IL-4 for an additional 3 days. Finally, the cells were harvested, washed and stimulated with PMA (Sigma, Cat. No. P-8139, 5 ng/mL) and ionomycin (Sigma, Cat. No. I-0634, 500 ng/mL) in the presence of GolgiStop™ (Cat. No. 554724) for 4 hr at 37 °C. The activated cells were harvested and the presence of TNF producing cells was detected by immunocytochemistry using a three-step staining procedure that employs a Biotin Goat anti-Mouse IgG secondary antibody (Cat. No. 550337) and a horseradish peroxidase-based detection system (Streptavidin-HRP Cat. No. 550947) (Nomarski optics, original magnification 400X). To demonstrate specificity of staining the binding of the MAb11 (Cat. No. 559071) antibody was blocked by the preincubation of the purified antibody with excess recombinant human TNF protein (Cat. No. 554589, data not shown).

BD Pharmingen™ Purified Mouse Anti-Human TNF for ICC - Purified - Clone MAb11 - Isotype Mouse IgG1, κ - Reactivity Hu - 0.25 mg

Purification: The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.

Immunogène: Recombinant Human TNF

Clone: MAb11

Isotype: IgG1 kappa

Information d'application

Indications d'application: Optimal working dilution should be determined by the investigator.

Procédure de l'essai:

FOR IMMUNOCYTOCHEMICAL STAINING OF SINGLE-CELL PREPARATIONS This procedure describes the immunoenzymatic technique of staining cytokines within individual cells that are immobilized on microscopic slides via adherence (adherent slides) or centrifugation (cytospins). ADHESION SLIDES 1. Harvest cells and wash them twice in PBS using centrifugation (400 x g for 5 min) to remove residual protein. 2. Adjust the cell concentration at 4-5 x 10e6 cells/mL in PBS. 3. Place 20 μL of the cell suspension in each well of the adhesion slides and let them adhere at room temperature (RT) for 20 min. Please note that the slides should be washed in PBS at RT for 5 min before transferring the cells. 4. Fix cells on slides using fixation buffer for 15 min at RT. 5. Wash slides 2X in PBS with 5 min incubations. 6. Block slides with PBS supplemented with 1 % (w/v) BSA (Sigma, Cat. No. A43-78) for 30 min at RT or 10 min at 37 °C. 7. Wash slides 2X in PBS and proceed with staining or air dry them and store them at -80 °C for future use. 8. Incubate slides with 20 μL of 1 % goat serum and PBS with 0.1 % (w/v) saponin for 30 min at RT. 9. Wash slides 2X with PBS with 5 min incubations. 10. Block endogenous peroxidase activity with Endogenous Peroxidase Blocking Buffer (20 μL/well) for 10 min at RT. 11. Wash 2X in PBS with 5 min incubations. 12. Incubate each well with Avidin (20 μL/well) for 15 min. 13. Wash 2X in PBS with 5 min incubations. 14. Incubate each well with Biotin (20 μL/well) for 15 min. 15. Wash 2X in PBS with 5 min incubations. 16. Incubate each well for 1 hr at RT with 20 μL of purified cytokine-specific antibody or appropriate immunoglobulin isotype control diluted in IHC Diluent Buffer (Cat. No. 559148) supplemented with saponin. 17. Wash slides 2X in PBS with 5 min incubations. 18. Incubate each well with 20 μL of a biotinylated secondary antibody diluted in IHC Diluent Buffer for 30 min at RT. 19. Wash 2X in PBS with 5 min incubations. 20. Apply 20 μL of Streptavidin-HRP (BD Cat. No. 550946) to each well on slides and incubate for 30 min at RT. 21. Wash slides 2X with PBS with 5 minutes incubations. 22. Incubate with DAB Substrate as directed, (BD Cat. No. 550880) for less than 5 min at RT. 23. Stop the development of the color reaction by washing with PBS. 24. The slides are subsequently mounted in short-term storage mounting medium. CYTOSPINS 1. Assemble the Cytospin's sample chamber (e.g. Cytospin 3, Shandon, UK or comparable centrifuge), filter card, slide and cytospin racks according to manufacturer's specifications. 2. Load 40 μL of approximately 1 x 10e6 cells to each sample chamber. 3. Spin slides at 600 rpm for 2 min. 4. Take slides out of the cytospin rack and place them on a staining rack. 5. For fixation and staining please follow the steps 4 through 24 specified above for staining cells on adhesion slides.

Restrictions: For Research Use only

Stockage

Concentration: 0.5 mg/mL

Buffer: Aqueous buffered solution containing ≤0.09 % sodium azide.

Agent conservateur: Sodium azide

Précaution d'utilisation: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Stock: 4 °C

Stockage commentaire: Store undiluted at 4°C.

Références pour anticorps anti-TNF alpha (ABIN2689935)

Rathjen, Cowan, Furphy, Aston: "Antigenic structure of human tumour necrosis factor: recognition of distinct regions of TNF alpha by different tumour cell receptors." dans: Molecular immunology, Vol. 28, Issue 1-2, pp. 79-86, (1991) (PubMed).

Hsu, Raine, Fanger: "Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures." dans: The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, Vol. 29, Issue 4, pp. 577-80, (1981) (PubMed).

Détails sur TNF alpha

Antigène: TNF alpha (Tumor Necrosis Factor alpha (TNF alpha))

Autre désignation: TNF (TNF alpha Produits)

Synonymes: anticorps DIF, anticorps TNF-alpha, anticorps TNFA, anticorps TNFSF2, anticorps RATTNF, anticorps Tnfa, anticorps tnf, anticorps TNF-a, anticorps TNFalpha, anticorps Tnfsf1a, anticorps TNFa, anticorps cTNF, anticorps Tnf-alpha, anticorps tnfa-like, anticorps TNF-ALPHA, anticorps dif, anticorps tnfa, anticorps xtnf, anticorps tnfsf2, anticorps tnf-alpha, anticorps Cachectin, anticorps tumor necrosis factor, anticorps tumor necrosis factor b (TNF superfamily, member 2), anticorps tumor necrosis factor alpha, anticorps tumor necrosis factor a (TNF superfamily, member 2), anticorps TNF, anticorps Tnf, anticorps tnf, anticorps tnfb, anticorps tnf-alpha, anticorps LOC103694380, anticorps tnfa

Pathways: Signalisation NF-kappaB, Apoptose, Caspase Cascade in Apoptosis, Signalisation TLR, Cellular Response to Molecule of Bacterial Origin, Regulation of Leukocyte Mediated Immunity, Positive Regulation of Immune Effector Process, Production of Molecular Mediator of Immune Response, Positive Regulation of Endopeptidase Activity, Hepatitis C, Protein targeting to Nucleus, Inflammasome