IL-4 antibody

Catalog No. ABIN2689764

The Rat Monoclonal anti-IL-4 antibody has been validated for ICC. It is suitable to detect IL-4 in samples from Mouse.

Quick Overview for IL-4 antibody (ABIN2689764)

Target: IL-4 (IL4) (Interleukin 4 (IL4))

Reactivity: Mouse

Host: Rat

Clonality: Monoclonal

Conjugate: This IL-4 antibody is un-conjugated

Application: Immunocytochemistry (ICC)

Clone: 11B11

Product Details anti-IL-4 Antibody

Brand: BD Pharmingen™

Characteristics: The 11B11 antibody reacts with mouse interleukin-4 (IL-4). The immunogen used to generate the 11B11 hybridoma was partially purified mouse IL-4 from PMA-stimulated EL-4 supernatant. This is a neutralizing antibody. This antibody is routinely tested by immunocytochemical analysis. Other applications were tested during antibody development only or reported in the literature. RBC-lysed BALB/c splenocytes, were enriched in CD4+ cells by panning and were cultured for 2 days with plate bound anti-mouse CD3 and soluble anti-mouse anti-CD28 in the presence of recombinant mouse IL-2 and recombinant mouse IL-4. The cells were subsequently harvested, washed and recultured with recombinant mouse IL-2 and recombinant mouse IL-4 for an additional 3 days. Finally, the cells were harvested, washed and cultured with PMA (Sigma, Cat. #P 8139, 5 ng/mL) and ionomycin (Sigma, Cat. #I-0634, 500 ng/mL) in the presence of GolgiPlug™ (Cat. No. 555029) for 4 hr at 37 °C. The activated cells were harvested and the presence of IL-4 producing cells was detected by immunocytochemistry using a three-step staining procedure that employs a Biotin Goat anti-Rat IgG secondary antibody (Cat. No. 559286) and a horseradish peroxidase-based detection system. To demonstrate specificity of staining the binding of the 11B11 (Cat. No. 559602) antibody was blocked by the preincubation of the purified antibody with excess recombinant mouse IL-4 protein (Cat. No. 550067, data not shown). (Nomarski optics, original magnification 400 X).

BD Pharmingen™ Purified Rat Anti-Mouse IL-4 - Purified - Clone 11B11 - Isotype Rat IgG1 - Reactivity Ms - 0.25 mg

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

Immunogen: Partially Purified Mouse IL-4

Isotype: IgG1

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Assay 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 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 Antibody Diluent for IHC, 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 Antibody 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 mounding 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

Handling

Concentration: 0.5 mg/mL

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

Preservative: Sodium azide

Precaution of Use: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Storage: 4 °C

Storage Comment: Store undiluted at 4°C.

Target Details for IL-4

Target: IL-4 (IL4) (Interleukin 4 (IL4))

Alternative Name: IL-4

Pathways: JAK-STAT Signaling, Regulation of Leukocyte Mediated Immunity, Positive Regulation of Immune Effector Process, Production of Molecular Mediator of Immune Response, Proton Transport, Activated T Cell Proliferation