Cells not sticking to chamber slides after fixing - (Feb/21/2008 )
hey guys, i am having some trouble with cells floating away after the fixing step. i am using PC12 celsl, which are supposed to be adherent. after i apply 4% paraformaldehyde for 10 minutes, i noticed that some cell clumps begain to float away during the PBS wash step. is it the paraformaldehyde that is not working? thanks.
You might have to use PBS with calcium and magnesium. This helps the cells to stick better to the plates during immunochemistry.
I used to fix with paraformaldehyde for 20 minutes. Maybe you don`t fix your cells long enough??
Have fixed cells with 2% PFA in PBS, and they remained sticky (less than 10 minutes should be fine btw). Only thing that comes to mind is calcium/magnesium. Do your cells come off if you apply just PBS to them?
the funny thing is the cells look fine in the very first wash steps when i remove the culture medium. after fixing, and doing some more washes, it's when i notice the cells are not stuck to the surface at all. by the time i get to the permeabilization step, there are hardly any cells left at all. the cells do look healthy, however.
My cells used to fine till the wash after the primary antibody and then I would lose them and byt the time I had to mount them , I would be lucky to get a small strip of cells attached to the coverslip.
have you tried adding the fixing stuff very *very* slowly to the cells.
my cells just didn't like it, and would slide right off, until i became really careful and added the stuff slowly. be delicate.
V
how much magnesium and/or calcium do you add? our lab makes up our own PBS.
how much magnesium and/or calcium do you add? our lab makes up our own PBS.
we buy PBS with calcium and magnesium as 10x from invitrogen and then make a 1x PBS. Its expensive, so we only use it for immunostaining.
this if from the invitrogen website. For additonal info, check it.
Calcium Chloride (CaCl2) (anhyd.): 100mg/L
Magnesium Chloride (MgCl2-6H2O): 100mg/L