ImmunofluorescenceofEndothelialCells内皮细胞免疫荧光.docVIP

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) Rinse 1X with ice cold EBM-2 (Basal media) and 1X with ice cold CMF-PBS Treat at room temperature for 10 minutes with room temperature Fixative Add equal volume of room temperature 2X Triton X-100 Solution additional 10 minutes Rinse 3X briefly with PBS Treat with Blocking solution for 10 minutes (very gentle shaking). (500 ul/well). 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). Rinse 4x10 minutes with PBS 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) Rinse as in step 7) 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). 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 Re