detection of vWF expression by immunostaining or immunofluorescence

 Recently I plan to characterize primary rat endothelial cells by immunological methods. Thus I ordered vWF antibody from Abcam (ab6994) to detect the vWF expression in endothelial cells. Before the experiment, I selected MS1, a pancreas islet endothelial cell line, and the 293, respectively as the positive and negative controls.

I used the ABC method for immunohistochemistry and the indirect fluoresent antibody method for immuofluorescence. For both of these methods, weak positive signal was detected for the MS1 endothelial cells, and no signal was detected for this cell line with only PBS added. Significantly, weak positive signal was also detected for 293 cells.

These results really bewildered me. I wonder what factor resulted in weak positive signal for the MS1 cells. In the introduction of the Abcam vWF antibody, it was used for detected of murine vWF (MS1 is a murine cell line) but the result was not consistent. 293 cells have been thought without vWF expression.

Thus I wonder if problem came from the antibody or the endothelial cells. Any advice on interpretation of these results and the correct methodology for characterization of primary endothelial cells is welcome.

Hi yanglin0,

Have you also included secondary antibody alone? It could be that the condition of the fluorescent conjugated secondary Ab is not optimal.

Good luck.

Hi Pippuri,

I did set up one control in which only PBS and secondary antibody was added. No fluorescent signal was detected in this well. Thus there seemed not to be background from the secondary antibody.

Furthermore, I used the vWF antibody to detect vWF antibody expression in endothelial cells and 293 cells (please see the attached file). Weak positive signal was detected in the endothelial cell lane but non-specific bands were detected both in the endothelial and 293 cell lanes. From left to the right were the protein markers, endothelial and 293 cell lanes.

Thus I am going to change the vWF antibody for the further experiments. Please let me know if you have any other comments.

Thanks