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BD Pharmingen™ Purified Rat Anti-Mouse IFN-γ
Clone XMG1.2 (RUO)
Immunocytochemistry analysis of IFN-γ expression on mouse splenocytes. RBC-lysed BALB/c splenocytes were cultured with PMA (Sigma, Cat. No. P-8139, 5 ng/ml) and ionomycin (Sigma, 500 ng/ml, Cat. No. I-0634) and GolgiPlug™ (BD Cat. No. 555029) for 4 hours at 37°C. The activated cells were harvested and the level of IFN-γ producing cells were 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 400X).
Immunocytochemistry analysis of IFN-γ expression on mouse splenocytes. RBC-lysed BALB/c splenocytes were cultured with PMA (Sigma, Cat. No. P-8139, 5 ng/ml) and ionomycin (Sigma, 500 ng/ml, Cat. No. I-0634) and GolgiPlug™ (BD Cat. No. 555029) for 4 hours at 37°C. The activated cells were harvested and the level of IFN-γ producing cells were 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 400X).
Product Details
BD Pharmingen™
Ifg; Ifng; IFN-γ; IFN-g; IFN-gamma; Interferon gamma; Type II Interferon
Mouse (QC Testing)
Rat IgG1, κ
Mouse IFN-γ Recombinant Protein
ELISA (Routinely Tested), Immunocytochemistry (Tested During Development)
0.5 mg/ml
AB_2123177
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: Purified Rat Anti-Mouse IFN-γ (Cat. No. 559065) antibody can be used to identify and enumerate human IFN-γ 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)
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™ Cytokine ICC Diluent Buffer supplemented with saponin (BD Cat. No. 550009).
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
1. Biotin Goat anti-Rat IgG (Cat. No. 559286), or 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 Pharmingen's Cytokine ICC Diluent Buffer 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 ICC Cytokine 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.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- 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.
- 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.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Purified Rat IgG1, κ Isotype Control RUO
Size
0.25 mg
Cat No.
559072
Streptavidin HRP RUO
Size
50 ML
Cat No.
550946
Anti-Rat Ig HRP Detection Kit RUO
Size
200 Tests
Cat No.
551013
Biotin Goat Anti-Rat Ig RUO
Size
0.5 mg
Cat No.
559286
Protein Transport Inhibitor (Containing Brefeldin A) RUO
Size
1 ML
Cat No.
555029
559065 Rev. 2
Antibody Details
XMG1.2
The XMG1.2 monoclonal antibody specifically binds to mouse interferon-γ (IFN-γ) protein. IFN-γ is a pleiotropic cytokine, of approximately 15-17 kDa, involved in the regulation of inflammatory and immune responses. It plays an important role in activation, growth, and differentiation of T and B lymphocytes, macrophages, NK cells and other non-hematopoietic cell types. IFN-γ production is associated with the Th1 cell differentiation. The purified form of this antibody has been reported to be a neutralizing antibody.
559065 Rev. 2
Format Details
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
559065 Rev.2
Citations & References
View product citations for antibody "559065" on CiteAb
Development References (5)
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Abrams JS, Roncarolo MG, Yssel H, Andersson U, Gleich GJ, Silver JE. Strategies of anti-cytokine monoclonal antibody development: immunoassay of IL-10 and IL-5 in clinical samples. Immunol Rev. 1992; 127:5-24. (Clone-specific). View Reference
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Cherwinski HM, Schumacher JH, Brown KD, Mosmann TR. Two types of mouse helper T cell clone. III. Further differences in lymphokine synthesis between Th1 and Th2 clones revealed by RNA hybridization, functionally monospecific bioassays, and monoclonal antibodies. J Exp Med. 1987; 166(5):1229-1244. (Clone-specific). View Reference
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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
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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
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Sander B, Hoiden I, Andersson U, Moller E, Abrams JS. Similar frequencies and kinetics of cytokine producing cells in murine peripheral blood and spleen. Cytokine detection by immunoassay and intracellular immunostaining. J Immunol Methods. 1993; 166(2):201-214. (Clone-specific: Immunocytochemistry (cytospins)). View Reference
559065 Rev. 2
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.