Hi there,
I am trying to fix, stain, and image cells in mitosis with an antibody against gamma-H2AX. I do not synchronize my cells as part of my research looks at genome integrity, so I usually just grow them to a higher confluency and search for cells in metaphase or anaphase manually. I have been fixing with 4% formaldehyde at 37C for 10 minutes, and blocking with 5%BSA in TBS+0.01%Triton-X. I do this at 37C because I don't want the microtubules to depolymerize in response to cold fixative. However, I always seem to get non-specific background staining. There are a lot of little foci all over the place. I notice that they frequently colocalize with microtubules on the spindle, but I don't know why that would be happening. Would it be better if I used Ice cold methanol instead? Would this ruin any analysis I do on cells in metaphase in anaphase due to microtubule depolymerization?
Thanks!
Dear Noel,
In my experience, methanol minus 20C, 6 min is better for staining microtubules. Depolymerization of MT does not occur and no additional membrane permeabilization is required before immunofluorescence staining with antibodies. It is important that the methanol temperature is no warmer than minus 20C and its volume per cover glass is at least 10 ml. I usually prerared little bottles with fixative in the evening and left it in the freezer overnight. Before fixation, I rinsed the cells on the glass with warm PBS at 37 C, and removed a drop of PBS with a filter (on the side opposite to the cells) so that it did not get into the methanol. The main thing is to prepare everything in advance and act quickly. After fixing the coverslip with cells, they are washed with PBS at room temperature. Methanol should be taken with a minimum percentage of water.
Best regards,
Rustem Uzbekov
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