4 replies to this topic

[beny]

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

[bob1]

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

[beny]

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

[bob1]

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

[beny]

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