BD, 553443, BD Pharmingen™ FITC Rat Anti-Mouse IgG1

Alternative Name: Ighg1; Immunoglobulin heavy constant gamma 1; Igh-4
Reactivity: Mouse (QC Testing)
Isotype: Rat LOU, also known as Louvain, LOU/C, LOU/M IgG1, κ
Immunogen: Pooled Mouse IgG1
Application: Flow cytometry (Routinely Tested), Intracellular staining (flow cytometry) (Tested During Development)
Concentration: 0.5 mg/ml
RRID: AB_394862
Storage Buffer: Aqueous buffered solution containing protein stabilizer and ≤0.09% sodium azide.
Regulatory Status: RUO
Preparation And Storage: Preparation And Storage Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with FITC under optimum conditions, and unreacted FITC was removed.
Recommended Assay Procedures: Recommended Assay Procedures BD Pharmingen™ FITC Rat Anti-Mouse IgG1 antibody may be used as a primary or secondary reagent in immunofluorescent staining. IMMUNOFLUORESCENT STAINING OF INTRACELLULAR IMMUNOGLOBULIN (Ig) PROTOCOL 1.  Prepare a single-cell suspension and determine cell number. 2.  Suspend cells in staining buffer (PBS + 2% FBS + 0.1% Sodium Azide) at 2 x 10^7 cells/ml and transfer to U-bottom microwell plates in 50 µl/well for immunofluorescent staining. Note: The BD Pharmingen™ Stain Buffer with FBS (Cat. No. 554656) is effective for use as a staining buffer in this protocol. 3.  Block Fcγ receptors by adding 0.2 µg of Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block™) (Cat. No. 553141/553142) in 50 µl of staining buffer to each well. 4.  Incubate 5 minutes on ice. 5.  Add 200 µl of staining buffer/well and resuspend cells. Centrifuge at 250 x g for 5 minutes and aspirate supernatant. 6.  Block surface Ig with Purified Rat Anti-Mouse IgG1(Cat. No. 553440) by adding 1.0 µg per sample in 50 µl of staining buffer/well. Note: Surface markers may be stained during this step as described in the "Immunofluorescent Staining of Mouse and Rat Leukocytes for Flow Cytometry" in the Protocols section of our website under "Multicolor Flow" at http://www.bdbiosciences.com/us/s/resources. 7.  Incubate 15 minutes on ice. 8.  Wash 2x as described in Step 5. 9.   Resuspend cells in 100 µl of BD Cytofix/Cytoperm™ intracellular staining buffer (BD Cytofix/Cytoperm™ Kit, Cat. No. 554714) per well. 10. Incubate 30 minutes at room temperature. 11. Wash 2x with 200 µl of 1x Perm/Wash buffer (provided in the BD Cytofix/Cytoperm Kit) per well. Centrifuge at 250 x g for 5 minutes and aspirate supernatant between washes. 12. Stain intracellular Ig by adding ≤1 µg of BD Pharmingen™ FITC Rat Anti-Mouse IgG1( Cat. No. 553443/562026) in 50 µl of 1 x Perm/Wash buffer/well. Note: Other antibodies recommended for staining of intracellular markers may be added during this step as described in Step 12. 13. Incubate for 30 minutes at room temperature. 14. Wash 2x as described in Step 11. 15. Resuspend and transfer samples in 100 µl of staining buffer to tubes appropriate for analysis with a flow cytometer. Bring volume in each tube to 400 µl with staining buffer. 16. Analyze samples on a flow cytometer.