Immunofluorescence of frozen cell culture inserts - (Sep/06/2006 )
Hello 
I am currently working on 3-D cellculture of epithelial cells. They are successfully grown on inserts. And then the insert membrane is removed and cut as a frozen section in OCT.
My problem is when i come to do the immunofluorescence - I am having a problem in that i am losing most, if not all of the cells.
I think the problem lies in either the fixing, permeabilisation or blocking step. The cells are currently fixed in 4%PFA, permeablised in 0.1% Triton X in PBS, and blocked with 0.5% BSA with 5% Normal goat serum in PBS.
can anybody suggest anything to help- or even an alternative protocol.
all help very, very welcome.
Thank you -
L 
Have u tried using PBS with Calcium and Magnesium for washes and for making other solutions, as this can prevent cell loss during immunocytochemistry.
Hi
no i haven't , to be honest didn't think it would make that much difference -
i will give it a go and see if that helps - thank you
L
I agree with Scolix. We use PBS with Ca2+ and Mg2+ (PBS++) for our immunofluorescence and we have very little cell loss...
thank you both
just an addition - have either of you ever done this with cells grown on cell culture inserts set in OCT and then cut on cryostat?
was wondering if there is an optimum method of cutting the cells?
L
I am sorry, I have no clue abt cutting cells.
Tissues - yes, but cells - no .
Its ok - i usually only cut tissues - but the method is being addapted to cut cells.
and it is my luck to sort out the protocol! 
i am surei will have it all sorted soon enough - just if anyone had ever done it before and had any advise i would be most welcome!!
thanks for all the help.
forever frustrated - L 