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posted 07 Aug, 2006
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natural cytotoxicity receptors
Primary monoclonal mouse anti human antibodies (all obtained from R&D Systems, Inc. MN. USA): anti-human CXCR3 antibody (IgG1, clone49801, staining concentration of 10 micrograms/ml), anti-human CXCR1 mAb (IgG2A, clone 42705, staining concentration of 10 micrograms/ml), anti-human CXCR4 (IgG2A, clone 44708, staining concentration 10 micrograms/ml).
The mouse anti human mAb for HLA-G was previously described and kindly provided by Dr. Mike McMaster (mouse IgM, staining concentration of 10 micrograms/ml) .
1) Perform immunohistochemistry for chemokine receptors CXCR1, CXCR3 and CXCR4 on first trimester human placental paraffin-embedded sections as previously described , with microwave antigen retrieval using sodium citrate buffer (Zymed Laboratory Inc, San- Francisco, CA, USA).
2) Stain with the same concentration of isotype match controls for each chemokine receptor on the same serial sections as controls.
3) Perform immunohistochemistry on decidual sections with NCR-Fc fusion proteins (prepared as previously described 3) by microwave heating the formalin-fixed, paraffin embedded tissue sections in citrate buffer in order to retrieve antigens.
4) Then stain sections with the different NCR-Fc fusion proteins or control-Fc (8 µg/ml, final concentration) followed by biotinylated-goat-anti-human-Fc (Jackson ImmunoResearch, West Grove, PA).
5) Detect using the avidin-biotin peroxidase complex method with a Vectastain kit (Vector Laboratories, Burlingame, CA).