Fixing cells in wells? - (Mar/19/2008 )

Hello,

I know this may be a stupid question. I'm a newbie in doing immunocytochemical staining. I'm trying to fix cells on a cover slip in 6-well plates using PFA.

My question is, do i add the 3.5% PFA straight onto the cover slip IN the well after rinsing with PBS? Or do i remove the cover slip from the well first before i add the PFA on the cover slip?


I was confused over the protocol i have here.

Thanks a lot for the enlightenment!

I used to do all my staining on the well, and only removed the cover slips at the end (but I was using 24 wells plates, so volumes were much smaller).
I don't think it'll make a difference whether you fix them in or out of the well. just make sure you don't scratch the side where your cells are.

I usually fix my cell on coverslips in the well (12- or 24-well plate). Aspirate medium and add enough 3.7% formaldehyde to cover the coverslip. Fix at 4 degree Celsius for approx. 40 minutes. Then I do all the washing and staining in the well.

It is also possible to take the coverslip out and place it with cells down on parafilm (would do this after fixation though), this way you can save antibody.

the more often you remove a coverslip from the well the more likely you are to be staining the wrong side.

just do it in the well

btw - let the liquids run down the side of the well if possible to avoid washing off the cells

dom