Scientist Solutions - Partial adherent cell fixation

sentinel

Posted 8/9/2005 10:03:07 AM

Hi friends,

I am now doing with a cell line PU5-1.8, a marcophage like cell line. I want to fix it on cell culture treated coverslip and take to observe under microscope. But when I add mentanol to fix it, the cells turn to shrink and look ugly, just like a debris. Do anyone can give me some advice?

frasermoss

Posted 8/9/2005 3:23:45 PM

Why not use 0.02%-0.1% Triton X-100 instead (dissolved in your regular buffer e.g. PBS or TBS pH 7.4)? Incubate for about 15 min to get permableization.

Inclusion of small amounts of glutaraldehyde (0.1%) along with paraformaldehyde in the fixation step usually helps in preservation of cell structure and surface membrane proteins following permeabilisation by Triton X-100. In this case it may be necessary to determine if antigenicity is also maintained after glutaraldehyde is added.

If Triton X-100 is too harsh a detergent you could try saponin instead.

sentinel

Posted 8/10/2005 1:43:31 AM

Thanks to your suggestion first.

Fix with paraformaldehyde may be help, but if it compatible to staining solution? As I need to fix the cell and then stain the nucleus and cytoplasm. THX!

frasermoss

Posted 8/10/2005 3:22:37 PM

I always fixed with 4% paraformadahyde (in PBS or TBS) without problems. As long as you do the appropriate postitive and negative controls you should not have a problem either.

MadScientist74

Posted 8/30/2005 3:38:10 AM

frasermoss said:

I always fixed with 4% paraformadahyde (in PBS or TBS) without problems. As long as you do the appropriate postitive and negative controls you should not have a problem either.

Agreed.... 4% PFA has always worked for me too... you might need to rinse off the PFA/PBS with whatever buffer base you are using for the stain IF there is some reaction or irregular staining with the PBS by the staining reagent.

The cells shrink due to the methanol being too 'drying'...

Good luck!

tash

Posted 10/10/2005 6:33:03 AM

We use 4% PFA for all our ICC and don't seem to have any problems.

We did try methanol/acetone 1:1 at one point & that seemed to work fine as well (we were trying to troubleshoot a particular antibody that was giving us a headache).

I don't know if the addition of acetone would assist your situation, but might be worth a try if the PFA doesn't work.