I have been trying to fix some A431 cells in several 6-well plates, following the paraformaldehyde protocol step by step. Nonetheless, when I put the cells under the microscope, most of then were dead. I believe I have respected all the steps of this protocol, so I don't know what could have been wrong. I hope someone of you could help me if you have used this method before. Thank you very much.
You can try different protocols depending on the method you whan to use. For structure, the better fixatives are cold methanol or acetone/methanol. If you need to use paraformaldehyde, try to add the fixative slowly into the same medium you are using to culture the cells. Add it slowly until reaching the concentration of around 4% and let during 15-30 min.
Sorry to be flippant, but all your cells most certainly will be dead! I think you must mean that their morphology is different from when they were alive. If this is so, then contrary to what Guillermo suggested, I would wash your cells twice with PBS then fix with 4% paraformaldehyde in PBS for about 10 minutes, then wash again with fresh PBS. it is important to have the correct osmolarity in your fixative.
I usually grow my cells on sterile glass coverslips in 6-well plates if I am going to do immunocytochemistry on them. The whole procedure can then be done in the wells, including counterstaining. When you have finished, you dehydrate through an alcohol series, fish them out and dip 10 times in xylene and invert onto a drop of DPX on a clean glass slide.
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