IL-10 antibody

Catalog No. ABIN2689718

This Rat Monoclonal antibody specifically detects IL-10 in ICC. It exhibits reactivity toward Mouse and has been mentioned in 2+ publications.

Quick Overview for IL-10 antibody (ABIN2689718)

Target: IL-10 (IL10) (Interleukin 10 (IL10))

Reactivity: Mouse

Host: Rat

Clonality: Monoclonal

Conjugate: This IL-10 antibody is un-conjugated

Application: Immunocytochemistry (ICC)

Clone: JES5

Product Details anti-IL-10 Antibody

Brand: BD Pharmingen™

Characteristics: The JES5-16E3 antibody reacts with mouse interleukin-10 (IL-10). The immunogen used to generate the JES5-16E3 hybridoma was recombinant mouse IL-10. This is a neutralizing antibody. This antibody is routinely tested by immunocytochemistry. Other applications were tested during antibody development or reported in the literature. Immunocytochemistry on stimulated cells. 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, 5 ng/mL) and ionomycin (Sigma, 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-10 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 (Cat. No. 550946) (Nomarski optics, original magnification 400 X). To demonstrate specificity of staining the binding of the JES5-16E3 (Cat. No. 559063) antibody was blocked by the preincubation of the purified antibody with excess recombinant mouse IL-10 protein (Cat. No. 550070, data not shown).

BD Pharmingen™ Purified Rat Anti-Mouse IL-10 - Purified - Clone JES5-16E3 - Isotype Rat IgG2b - Reactivity Ms - 0.25 mg

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

Immunogen: Recombinant mouse IL-10

Isotype: IgG2b

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 BD Pharmingen 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 with saponin 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

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.

References for anti-IL-10 antibody (ABIN2689718)

Litton, Sander, Murphy, OGarra, Abrams: "Early expression of cytokines in lymph nodes after treatment in vivo with Staphylococcus enterotoxin B." in: Journal of immunological methods, Vol. 175, Issue 1, pp. 47-58, (1994) (PubMed).

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

Target Details for IL-10

Target: IL-10 (IL10) (Interleukin 10 (IL10))

Alternative Name: IL-10

Pathways: Cellular Response to Molecule of Bacterial Origin, Regulation of Leukocyte Mediated Immunity, Production of Molecular Mediator of Immune Response, Maintenance of Protein Location, Cancer Immune Checkpoints