cell lysis - confocal slides (Jun/10/2011 )

hi friends
i am facing this problem for quiet long.
when even i make slides for confocal and go for analysis my cell lyse.
first i do all my experiment on tubes as they are suspension culture.
3 % paraformaldehyde fixation and keep it in 4oC till i collect all my samples.
then renationalisation with methanol at -20 for 10 min or with triton x 100 0.5 % 10 min 4 oC
then blocking followed by antibody treatment.
then i wash with PBS and add 8ul of mounting media on slide and over it keep 20ul of cell suspension.
i check in microscope they are fine .will keep the coverslip on top of it and seal it.check them again cell are ok .but if the same thing i check after 1 hour cell get lysed.
what can be the reason .
please help its getting into my nerves.
Beny

How long are you keeping the cells. Fixing and storing at 4 deg C in PBS will only work for a week or so.

i am keeping for only 2-3 days...
i think the cell are getting crushed when i put the coverslip.
how can i avoid that

They shouldn't be crushed by the coverslip, unless you happen to slip and cause the coverslip to move sideways while pressing down.

as the cells are suspension cells when i keep the coverslip it slides a little bit while sealing it .
how can i avoid that is there any other way to keep the coverslip so that it doesn't slide