I am trying to fix cells that were growing on a cover slip/glass slide. These are adherent epithelial cells. I did some research and found that a formalin, Glutaraldheyde and PBS combo works. I tried it but my cells don't seem to like it. Any suggestions ?
Thanks Philipp ..mine r epithelial and yes I will use them for microscopy imaging and not really for staining. I coated with poly lysine and so far its working so now I am figuring out ways for fixing them so I can store long while using for microscopy!
I use 4% PFA 10-15 minutes at RT for MEFs and Macrophages.
It is working for both cell types
make sure that all your medium is washed away.
Wash cells twice with PBS to remove medium. Fix in 4% paraformaldehyde for 10-15 mins at RT. Wash twice more with PBS. Permeablise with 0.5% Triton in PBS if required (usually only for staining). Wash again and carry on with required procedure. I know you stated that you're not going to stain the cells but if you decide to later, be careful with coating the coverslips as this can increase background fluorescence!
@ Julien.. haha yes. what I meant was they can't be really seen under confocal. I am assuming if that is becoz cells could not handle that chemical combo I mentioned earlier.
@ Phillipp I used poly D lysine and yes I washed with PBS. I will try PFA as you guys suggested.
Did anyone had any experience with glutaraldehyde alone ? - I heard that it has slow penetration and will be less toxic to cells
HI RT-P,
I normally incubate FetalBovineSerum onto cover slips before seeding epithelial cells - they seem to grow quite well like that? Also, I have had adherance problems in the past with PBS washing so certainly try it with additional Mg or Ca, but also with serum-free growth media if you can afford to.
I also fix cells for microscopy - 4% fomalin is best for confocal due to the auto fluorescence of glutaraldehyde, but the latter is better for electron microscopy if you are fixing for SEM?
I hope that helps!
Same as others, do a 4% PFA fixation after having washing media off my cells with two PBS rinse. I do not if it may help but everything is ice-cold or straight from the fridge and have the plate incubating with the PFA over some ice. Then I proceed with the immunostaining.
My take? Find the right coating (I use gelatin in glass included in a 24-well plate), chill out (fixation at 4ºC) and be extra careful and gentle with everything you add in and remove.
1. Coat the coverslip with poly-L-Lysine and grow cells on it.
2. 3% Formaldehyde + 0.1% glutraldehyde in PBS can be used for fixing the cells. Intial wash the cells twice with PBS and then fix the cells using solution above for 10 min followed by thrice washing with PBS again. store in PBS at 4 degree.
Thank you all.. I will try with PFA first as we have that avaialble!
@ Mark - How long did you treat with 4% formalin? also is it buffered with PBS? The formalin we bought consists of formaldehyde in methanol- so wondering if that affects the fixation!
Methanol is a fixative itself (often used in conjunction with acetone), so it's presence will probably affect your results, unless it's at a very low concentration. Methanol and other organic solvents will remove lipids from membranes and usually destroy the structure of membranes, so keep that in mind if it's important for your later analysis. Alcohol fixation is generally used when you are interested in cytoskeletal structures.
Oh i C! I am actually looking for some toxicity of chemical. Since we are not sure whats going on inside cell as of now.. I think it would be nice to choose fixative that doesn't destroy any cellular structures
I agree to use 4% PFA. I use same procedure in lab work. But I use 0.01% triton X-100 for permeabilisation.
I think that the answers from Eric Patterson and Amit are good.
You could also try to use methanol:acetic acid 3:1 which it is used to fix cell for micronucleus... an advice... use cold solutions!
Coating the coverslip with poly-L-Lysine can be an help to ensure that cells are not removed by washing!
Hi RtP
If you are going to study the toxicity of some chemicals on your cells, i suggest to remove media, wash cells once with 1X PBS and fix your cells by 10% neutral buffer formalin for 10 minutes. Then, wash again with PBS for ten minutes. Finally, i suggest to use chamber slide instead of cover slips.
Good luck
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