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Purified Rat Anti-Mouse IL-4
Purified Rat Anti-Mouse IL-4

Immunocytochemistry analysis of IL-4 expression on mouse splenocytes. RBC-lysed BALB/c splenocytes were enriched for CD4+ cells by panning and cultured for 2 days with plate-bound Purified NA/LE Hamster Anti-Mouse CD3e (Cat. No. 553057; 10 λg/ml for coating) and soluble Purified NA/LE Hamster Anti-Mouse CD28 ( Cat. No. 553294; 2 λg/ml) in the presence of Recombinant IL-2 ( Cat. No. 550069; 10 ng/ml) and IL-4 (Cat. No. 550067; 25 ng/ml). The cells were subsequently harvested, washed and recultured with mouse IL-2 and 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 Purified Rat Anti-Mouse IL-4 (Cat. No. 559062) antibody was blocked by the preincubation of the purified antibody with excess recombinant mouse IL-4 protein (data not shown). (Nomarski optics, original magnification 400 X).

Immunocytochemistry analysis of IL-4 expression on mouse splenocytes. RBC-lysed BALB/c splenocytes were enriched for CD4+ cells by panning and cultured for 2 days with plate-bound Purified NA/LE Hamster Anti-Mouse CD3e (Cat. No. 553057; 10 λg/ml for coating) and soluble Purified NA/LE Hamster Anti-Mouse CD28 ( Cat. No. 553294; 2 λg/ml) in the presence of Recombinant IL-2 ( Cat. No. 550069; 10 ng/ml) and IL-4 (Cat. No. 550067; 25 ng/ml). The cells were subsequently harvested, washed and recultured with mouse IL-2 and 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 Purified Rat Anti-Mouse IL-4 (Cat. No. 559062) antibody was blocked by the preincubation of the purified antibody with excess recombinant mouse IL-4 protein (data not shown). (Nomarski optics, original magnification 400 X).

Product Details
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BD Pharmingen™
IL4: Interleukin-4; BSF-1; B-cell growth factor 1; BCGF-1
Mouse (QC Testing)
Rat IgG1
Partially Purified Mouse IL-4
ELISA (Routinely Tested), Immunocytochemistry (Tested During Development)
0.5 mg/ml
AB_397187
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO


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

Immunocytochemistry:

This antibody can be used to identify and enumerate human IL-4 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 the immunocytochemical procedure.  The avidin/biotin method is a highly sensitive method, because it employs a mixture of avidin and biotinylated enzyme complexes to increase immunoenzymatic signals. For optimal detection of cytokine producing cells, horseradish peroxidase is the preferred enzyme system.

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™ Antibody Diluent for IHC, Cat. No. 559148, supplemented with saponin

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-Rat Ig HRP Detection Kit (Cat. No. 551013).

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-Rat Ig HRP Detection Kit (Cat. No. 551013).

SECONDARY ANTIBODIES

Biotin Goat anti-Rat IgG (Cat. No. 559286) or Anti-Rat Ig HRP Detection Kit (Cat. No. 551013).

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.

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  3. Sodium azide is a reversible inhibitor of oxidative metabolism; therefore, antibody preparations containing this preservative agent must not be used in cell cultures nor injected into animals. Sodium azide may be removed by washing stained cells or plate-bound antibody or dialyzing soluble antibody in sodium azide-free buffer. Since endotoxin may also affect the results of functional studies, we recommend the NA/LE (No Azide/Low Endotoxin) antibody format, if available, for in vitro and in vivo use.
  4. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
559062 Rev. 3
Antibody Details
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11B11

Interleukin-4 (IL-4) is a pleiotropic cytokine that has many roles, such as inducing the differentiation of naïve helper T cells (Th0 cells) to Th2 cells, stimulating activated B-cell and T-cell proliferation, and promoting immunoglobulin class switching to IgG1 and IgE in mouse B-cells.  IL-4 is expressed by CD4 T-cells, mast cells, basophils and eosinophils.  IL-4 was previously known as B-Cell Differentiation Factor (BCDF) or B-cell Stimulatory Factor (BSF1).  The 11B11 monoclonal antibody specifically binds to mouse IL-4. The immunogen used to generate the 11B11 hybridoma was partially purified mouse IL-4 prepared from the supernatant of Phorbol 12-Myristate 13-Acetate (PMA)-stimulated EL-4 cells. The 11B11 antibody is reportedly a neutralizing antibody.

559062 Rev. 3
Format Details
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Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
559062 Rev.3
Citations & References
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Development References (7)

  1. Abrams J. Immunoenzymetric assay of mouse and human cytokines using NIP-labeled anti-cytokine antibodies. Curr Protoc Immunol. 2001; 1:6.20-6.21. (Clone-specific: ELISA). View Reference
  2. Hsu SM, Raine L, Fanger H. A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Am J Clin Pathol. 1981; 75(5):734-738. (Methodology: Immunocytochemistry (cytospins)). View Reference
  3. Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem. 1981; 29(4):577-580. (Methodology: Immunocytochemistry (cytospins)). View Reference
  4. Lindqvist C, Lundstrom H, Oker-Blom C, Akerman KE. Enhanced IL-4-mediated D10.G4.1 proliferation with suboptimal concentrations of anti-IL-4 receptor monoclonal antibodies. J Immunol. 1993; 150(2):394-398. (Clone-specific: Neutralization). View Reference
  5. Ohara J, Paul WE. Production of a monoclonal antibody to and molecular characterization of B-cell stimulatory factor-1. Nature. 1985; 315(6017):333-336. (Immunogen). View Reference
  6. Sander B, Andersson J, Andersson U. Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure. Immunol Rev. 1991; 119:65-93. (Clone-specific: Immunocytochemistry (cytospins)). View Reference
  7. Swain SL, Weinberg AD, English M, Huston G. IL-4 directs the development of Th2-like helper effectors. J Immunol. 1990; 145(11):3796-3806. (Clone-specific: Neutralization). View Reference
View All (7) View Less
559062 Rev. 3

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For Research Use Only. Not for use in diagnostic or therapeutic procedures.