T lymphocytes fixation on microscope slide! - For lipid rafts polarization localisation (Apr/13/2007 )

Hello all,

I need to do a fixation and a permeabilization of T lymphocytes on a microscope slide. I have a protocol to process, but I am not sure it's the right way...

I have to wash my cells with PBS and resuspend them in few microliters and put 4 ul on a microscope preliminary slide wahed with ethanol. I have to let dry for 15 minutes on the slide. I have to fix it 5 minutes in a bath of paraformaldehyde 2%-PBS and wash three times 5 minutes in PBS. I am not sure if the cells will still be attached to the slide. Do someone done something like that before? Do you have better idea?

Thanks a lot for your help!

Have a nice week-end!

Brassap

you can do it on a polylysine coated slide

strongly positively charged, for cells to adhere.
I never tried a comparison however.
I can only tell you it was working fine. Just let the cells sediment and they stick.

I would say it should work ? never tried.
by the way : I label, fix with PFA and then I stick the cell on the slide.

I incubate the cells in 96 wells, V-bottom wells, with fluorochrome conjugated antibodies, in PBS BSA EDTA buffer.
wash, (centrifuge and discard supernatant by reverting the plate, like in ELISA), then I incubate in PBS plus PFA 1%, wash, resuspend the cell in PBS and put a drop of these cells on a polylysine coated slide. I let it 20 minutes so the cells deposit on the slide, and sticks on the polylysine. then I get rid of the PBS, add mountage medium and a cover.