Posted 10 June 2011 - 02:28 AM
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.
Posted 12 June 2011 - 03:56 PM
Posted 12 June 2011 - 08:56 PM
i think the cell are getting crushed when i put the coverslip.
how can i avoid that
Posted 13 June 2011 - 05:01 PM
Posted 14 June 2011 - 03:15 AM
how can i avoid that is there any other way to keep the coverslip so that it doesn't slide