Immunofluorescence of Endothelial Cells



Immunofluorescence of Endothelial Cells

Materials:

Mounting Media: Prolong™ from Molecular Probes (Eugene, OR).

10X PBS plus Ca++/Mg++:

For 1X PBS plus Ca++/Mg++: Add 100mL of Solution A + B to 500 mL H2O and pH to 7.4 if needed

While stirring, slowly add 100mL of Solution C

Fill to 1 Liter (Do not heat/autoclave when Ca++/Mg++ are present)

Fixative:

2X Triton X-100:

Blocking Solution:

*Goat serum is added to enhance monoclonal antibody binding specificity. Do not use goat serum for polyclonal antibodies (since many pAb are generated in goats).

HMVEC-L (Clonetics)

1) Rinse 1X with ice cold EBM-2 (Basal media) and 1X with ice cold CMF-PBS

2) Treat at room temperature for 10 minutes with room temperature Fixative

3) Add equal volume of room temperature 2X Triton X-100 Solution additional 10 minutes

4) Rinse 3X briefly with PBS

5) Treat with Blocking solution for 10 minutes (very gentle shaking). (500 ul/well).

6) Treat with primary antibody** diluted roughly 1:10 to 1:100 in Blocking Solution for 45 minutes. (250 ul/well of a 2-well cultureslide, 150 ul/well of an 8-well cultureslide).

7) Rinse 4x10 minutes with PBS

8) Treat with secondary antibody labeled with Oregon Green 488 (Molecular Probes) diluted 1:50 in Blocking Solution for 30 minutes. (Add 735 ul blocking solution to 15 ul OG stock)

9) Rinse as in step 7)

10) Use Molecular Probe’s ProLong kit according to manufacturer’s instructions. (Add 1 ml of solution B to vial A, pipet to mix, add a drop of mixed mounting media on the slide, place coverslip on, seal coverslip with nail polish).

11) Store slides at –20oC and protected from light.

**If staining for F-actin, use 1U/coverslip of Rhodamine-Phalloidin (Molecular Probes). Prepare solution by diluting stock 200 Unit/ml solution 1:50 in blocking solution (add 1.5 ml blocking solution in 30 ul stock R-P). If double labeling, primary antibody can be diluted in the R-P solution. Subsequently add OG secondary antibody to bind the primary antibody. Do not use Texas Red secondary antibodies with R-P (same fluorescence).

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|Solution A +B (in order of listing) |

|To 800 mL H2O add: |

|2.0 g/L KH2PO4 |

|11.41g/L Na2HPO4 |

|pH to 7.4 |

|80.0 g/L NaCl |

|2.0 g/L KCl |

|Fill to 1 Liter |

|Solution C |

|1.057 g/L CaCl2 Anhydrous |

|0.469 g/L MgCl2 Anhydrous |

|To a 100mL glass bottle add: |

|3.7g Paraformaldehyde powder (Final 3.7% (w/v)) |

|80 mL of H2O |

|Heat in 85(C Water bath to dissolve (Add a couple of drops of 1N NaOH if needed) |

|After cooling, add 10mL of 10X PBS Solution A + B |

|Slowly add 10mL of 10X PBS solution C |

|Add 250(L of 50% aqueous glutaraldehyde (Final 0.125%) |

*Check final pH with litmus paper, adjust to 7.4 if needed

**Best if made fresh; can store about 1 week at +4(C

|To 10mL of 1X PBS add: |

|0.1g BSA (Final 1%) |

|100(L Normal Goat Serum (Cappel; reconstitute from powder) (1% Final) |

|To 50mL of Fixative: |

|200(L Triton X-100 (Final 1X Concentration 0.2%) |

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