Thanks Parvaiz. Let me briefly describe objective of my experiment. I would treat endothelial cells with thrombin or VEGF and with time want to monitor the internalization of VE Cashed in through confocal microscopy. I would use primary antibody for VE Cad and secondary with a fluorescent tag like FITC or Alexaflour 488. I need a protocol that can tell how to fix my cells, how to permeabilise and when. Because I would get signal from both cell surface and from within the cell.

Hope to get response.

Use a gfp tagged protein.

Hello Shadab,

I used the anti-TLR4 primary antibody and secondary antibody IgG H&L (FITC labeled). Additionally, I used 4% formaldehyde (10-15 minutes) for fixation and 0.5% Triton-X100 for 15 minutes to permeabilize the cell. The cells were washed three times after every step with PBS-T followed by confocal microscopy. As of the time period, it will solely depend on your experimental design and cell-line. Therefore, you will have to standardize the period of experiment in time dependent-manner as Mohammad Parvaiz Qamar Farshori

Best wishes,

Mahmudul Hasan