MHC Class II RT1D Mouse Monoclonal Antibody [Clone ID: OX-17]

CAT#: CL121B

MHC Class II RT1D mouse monoclonal antibody, clone OX-17, Biotin

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Specifications

Product Data

• Clone Name: OX-17
• Applications: FC, IHC
• Recommended Dilution: Flow Cytometry.
  Immunohistochemistry on frozen sections.
• Reactivities: Rat
• Host: Mouse
• Isotype: IgG1
• Clonality: Monoclonal
• Immunogen: Rat spleen membrane glycoproteins depleted of Ia-A antigens.
  Immunocyte Donor: BALB/c spleen
  Fusion Partner: X63 Ag8.653
• Specificity: This monoclonal antibody recognizes a monomorphic determinant on the a chain of the rat Ia antigen and appears to be the rat homologue of mouse Ia-E. It recognizes the rat Ia product present on B, but not T cells from lymph node or thoracic duct lymph. It does not bind to thymocytes or erythrocytes. The antibody does not cross-react with rat Ia-A or mouse Ia-E antigen, but rabbit antibody raised against the antibody affinity column-purified MRC OX-17 antigen cross-reacted on tissues of mice expressing Ia-E mouse antigen but not on those mouse strains that were Ia-E antigen negative.
• Formulation: PBS, 0.02% NaN3 and EIA grade BSA as a stabilizing protein to bring total protein concentration to 4-5 mg/ml
  Label: Biotin
  State: Liquid purified Ig
• Concentration: 0.1 mg/ml
• Purification: Protein G Chromatography
• Conjugation: Biotin
• Synonyms: HLA Class II
• Note: Protocol: FLOW CYTOMETRY ANALYSIS:
  
  Method:
  1. Prepare a cell suspension in media A. For cell preparations, deplete the red blood cell population with Lympholyte®-Rat cell separation medium.
  2. Wash 2 times.
  3. Resuspend the cells to a concentration of 2x10e7 cells/ml in media A. Add 50 µl of this suspension to each tube (each tube will then contain 1 x 10e6 cells, representing 1 test).
  4. To each tube, add 0.5-0.1 µg* of this Ab per 10e6 cells.
  5. Vortex the tubes to ensure thorough mixing of antibody and cells.
  6. Incubate the tubes for 30 minutes at 4°C.
  7. Wash 2 times at 4°C.
  8. Add 100 µl of secondary antibody (Streptavidin-FITC) at a 1:500 dilution.
  9. Incubate tubes at 4°C for 30 - 60 minutes (It is recommended that tubes are protected from light since most fluorochromes are light sensitive).
  10. Wash 2 times at 4°C.
  11. Resuspend the cell pellet in 50 µl ice cold media B.
  12. Transfer to suitable tubes for flow cytometric analysis containing 15 µl of propidium iodide at 0.5 mg/ml in PBS. This stains dead cells by intercalating in DNA.
  
  Media:
  A. Phosphate buffered saline (pH 7.2) + 5% normal serum of host species + sodium azide (100 µl of 2M sodium azide in 100 mls).
  B. Phosphate buffered saline (pH 7.2) + 0.5% Bovine serum albumin + sodium azide (100 µl of 2M sodium azide in 100 mls).
  
  Results - Tissue Distribution:
  Rat Strain: Fischer
  Cell Concentration: 1x10e6 cells per test
  Antibody Concentration Used: 0.1 µg/10e6 cells
  Isotypic Control: Biotin Mouse IgG1
  
  Cell Source Percentage of cells stained above control:
  Thymus 6.1%
  Spleen 48.8%
  Lymph Node 27.5%