BD, 559071, BD Pharmingen™ Purified Mouse Anti-Human TNF for ICC

Alternative Name: Tumor necrosis factor alpha; TNF-a; TNF-α; TNFSF2; Cachectin
Reactivity: Human (QC Testing), Rhesus, Cynomolgus, Baboon (Tested in Development)
Isotype: Mouse IgG1, κ
Immunogen: Recombinant Human TNF
Application: Intracellular block/flow cytometry (Routinely Tested)
Concentration: 0.5 mg/ml
RRID: AB_397191
Storage Buffer: Aqueous buffered solution containing ≤0.09% sodium azide.
Regulatory Status: RUO
Preparation And Storage: Preparation And Storage Store undiluted at 4°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
Recommended Assay Procedures: Recommended Assay Procedures Immunocytochemistry: Purified Mouse Anti-Human TNF for ICC (Cat. No. 559071) antibody can be used to identify and enumerate human TNF producing cells by immunocytochemistry. For optimal indirect immunocytochemical staining, the antibody should be titrated and visualized via a three-step staining procedure. Please see protocol below for a detailed description of our recommended procedure. CYTOKINE IMMUNOCYTOCHEMISTRY PROTOCOL REAGENTS REQUIRED 1. Fixation Buffer: 5% formalin (10% formalin, CMS, Cat. No. 245-684) is dissolved in phosphate buffered-saline (PBS) (Bacto  FA Buffer, Difco Laboratories, Cat. No.  2314-15-0), or BD Pharmingen™ ICC Fixation Buffer (BD Cat. No. 550010) 2. Endogenous Peroxidase Blocking Buffer: DAKO Peroxidase Blocking Reagent (DAKO, Cat. No. S2001). 3. Endogenous Biotin Blocking Buffer: Biotin/Avidin Blocking Kit (Vector Laboratories, Cat. No. SP-2001). 4. Antibody dilution buffer: BD Pharmingen™ IHC Antibody Diluent Buffer supplemented with saponin (Cat. No. 559148). 5. Microscopic slides: Adhesion Slides (Erie Scientific Company, Cat. No. ER-202B-AD) or for cytospins, Colorfrost /Plus slides (Fisher, Cat. No. 12-550-17). 6. Detection system: BD Pharmingen™ Streptavidin-horseradish peroxidase (HRP), (Cat. No. 550946), or Anti-Mouse Ig HRP Detection Kit (Cat. No. 551011). 7. Mounting medium for short-term storage: Aqua-mount®  (Lerner Laboratories, Cat. No. 13800). 8. DAB Substrate Kit (contains 3-3 -Diaminobenzidine tetra hydrochloride), (BD Cat. No. 550880), or Anti-Mouse Ig HRP Detection Kit (Cat. No. 551011). SECONDARY ANTIBODIES Biotin Goat anti-Mouse IgG (Cat. No. 550337) or Anti-Mouse Ig HRP Detection Kit (Cat. No. 551011) PROCEDURE 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 to 4-5 × 10×6 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 10×6 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.